The melano-macrophage: The black leukocyte of fish immunity.

Melanin and the process of melanin synthesis or melanogenesis have central roles in the immune system of insects, and production of melanin-synthesizing enzymes from their haemocytes may be induced following activation through danger signals. Melanin-containing macrophage-like cells have been extensively studied in amphibians and they are also present in reptiles. In fish, melano-macrophages are especially recognized with respect to melano-macrophage centres (MMCs), hypothesized to be analogues of germinal centres in secondary lymphoid organs of mammals and some birds. Melano-macrophages are in addition present in several inflammatory conditions, in particular melanised focal changes, or black spots, in the musculature of farmed Atlantic salmon, Salmo salar. Melanins are complex compounds that may be divided into different forms which all have the ability to absorb and scatter light. Other functions include the quenching of free radicals and a direct effect on the immune system. According to the common view held in the pigment cell community, vertebrate melanin synthesis with melanosome formation may only occur in cells of ectodermal origin. However, abundant information suggests that also myeloid cells of ectothermic vertebrates may be classified as melanocytes. Here, we discuss these opposing views and review relevant literature. Finally, we review the current status on the research concerning melanised focal muscle changes that represent the most severe quality problem in Norwegian salmon production, but also other diseases where melano-macrophages play important roles.

Persistent immune responses in the heart determine the outcome of cardiomyopathy syndrome in Atlantic salmon (Salmo salar).

Cardiomyopathy syndrome (CMS) caused by piscine myocarditis virus (PMCV) is a severe cardiac disease in Atlantic salmon (Salmo salar) and one of the leading causes of morbidity and mortality in the Norwegian aquaculture industry. Previous research suggest a variation in individual susceptibility to develop severe disease, however the role of the immune response in determining individual outcome of CMS is poorly understood particularly in cases where fish are also challenged by stress. The present study's aim was therefore to characterize cardiac transcriptional responses to PMCV infection in Atlantic salmon responding to infection under stressful conditions with a high versus low degree of histopathological damage. The study was performed as a large-scale controlled experiment of Atlantic salmon smolts from pre-challenge to 12 weeks post infection (wpi) with PMCV, during which fish were exposed to intermittent stressors. RNA sequencing (RNAseq) was used to compare the heart transcriptome of high responders (HR) with atrium histopathology score '4' and low responders (LR) with score '0.5' at 12 wpi. A high-throughput quantitative PCR (qPCR) analysis was used to compare immune gene transcription between individuals sampled at 6, 9 and 12 wpi. Based on RNAseq and qPCR results, RNAscope in situ hybridization (ISH) was performed for visualization of IFN-γ - and IFNb producing immune cells in affected heart tissue. Compared to LR, the transcription of 1592 genes was increased in HR at 12 wpi. Of these genes, around. 40 % were immune-related, including various chemokines, key antiviral response molecules, and genes. associated with a Th1 pro-inflammatory immune response. Further, the qPCR analysis confirmed. increased immune gene transcription in HR at both 9 and 12 wpi, despite a decrease in PMCV. transcription between these time points. Interestingly, increased IFNb transcription in HR suggests the. presence of high-quantity IFN secreting cells in the hearts of these individuals. Indeed, RNAscope. confirmed the presence of IFN-γ and IFNb-positive cells in the heart ventricle of HR but not LR. To conclude, our data indicate that in severe outcomes of PMCV infection various chemokines attract leucocytes to the salmon heart, including IFN-γ and IFNb-secreting cells, and that these cells play important roles in maintaining persistent antiviral responses and a sustained host immunopathology despite decreasing heart viral transcription.

A comprehensive transcriptional body map of Atlantic salmon unveils the vital role of the intestine in the immune system and highlights functional specialization within its compartments.

The intestine is a barrier organ that plays an important role in the immune system of Atlantic salmon. The immune functions are distributed among the diffuse gut lymphoid tissue containing diverse immune cells, and other cell types. Comparison of intestinal transcriptomes with those of other organs and tissues offers an opportunity to elucidate the specific roles of the intestine and its relationship with other parts of the body. In this work, a meta-analysis was performed on a large volume of data obtained using a genome-wide DNA oligonucleotide microarray. The intestine ranks third by the expression level of immune genes after the spleen and head kidney. The activity of antigen presentation and innate antiviral immunity is higher in the intestine than in any other tissue. By comparing transcriptome profiles, intestine shows the greatest similarity with the gill, head kidney, spleen, epidermis, and olfactory rosette (descending order), which emphasizes the integrity of the peripheral mucosal system and its strong connections with the major lymphoid organs. T cells-specific genes dominate among the genes co-expressed in these tissues. The transcription signature of CD8+ (86 genes, r > 0.9) includes a master gene of immune tolerance foxp3 and other negative regulators. Different segments of the intestine were compared in a separate experiment, in which expression gradients along the intestine were found across several functional groups of genes. The expression of luminal and intracellular (lysosome) proteases is markedly higher in pyloric caeca and distal intestine respectively. Steroid metabolism and cytochromes P450 are highly expressed in pyloric caeca and mid intestine while the distal intestine harbors genes related to vitamin and iron metabolism. The expression of genes for antigen presenting proteins and immunoglobulins shows a gradual increase towards the distal intestine.

Cell atlas of the Atlantic salmon spleen reveals immune cell heterogeneity and cell-specific responses to bacterial infection.

The spleen is a conserved secondary lymphoid organ that emerged in parallel to adaptive immunity in early jawed vertebrates. Recent studies have applied single cell transcriptomics to reveal the cellular composition of spleen in several species, cataloguing diverse immune cell types and subpopulations. In this study, 51,119 spleen nuclei transcriptomes were comprehensively investigated in the commercially important teleost Atlantic salmon (Salmo salar L.), contrasting control animals with those challenged with the bacterial pathogen Aeromonas salmonicida. We identified clusters of nuclei representing the expected major cell types, namely T cells, B cells, natural killer-like cells, granulocytes, mononuclear phagocytes, endothelial cells, mesenchymal cells, erythrocytes and thrombocytes. We discovered heterogeneity within several immune lineages, providing evidence for resident macrophages and melanomacrophages, infiltrating monocytes, several candidate dendritic cell subpopulations, and B cells at distinct stages of differentiation, including plasma cells and an igt + subset. We provide evidence for twelve candidate T cell subsets, including cd4+ T helper and regulatory T cells, one cd8+ subset, three γδT subsets, and populations double negative for cd4 and cd8. The number of genes showing differential expression during the early stages of Aeromonas infection was highly variable across immune cell types, with the largest changes observed in macrophages and infiltrating monocytes, followed by resting mature B cells. Our analysis provides evidence for a local inflammatory response to infection alongside B cell maturation in the spleen, and upregulation of ccr9 genes in igt + B cells, T helper and cd8+ cells, and monocytes, consistent with the recruitment of immune cell populations to the gut to deal with Aeromonas infection. Overall, this study provides a new cell-resolved perspective of the immune actions of Atlantic salmon spleen, highlighting extensive heterogeneity hidden to bulk transcriptomics. We further provide a large catalogue of cell-specific marker genes that can be leveraged to further explore the function and structural organization of the salmonid immune system.

Tumor-associated macrophages in canine visceral hemangiosarcoma.

Canine hemangiosarcoma (HSA) is a highly malignant tumor derived from hematopoietic stem cells and commonly occurs in visceral organs or skin. Visceral HSAs are particularly aggressive and progress rapidly despite multimodal treatment. Tumor-associated macrophages (TAMs) play a central role in carcinogenesis, tumor progression, and metastasis in humans and murine models. In this retrospective study, we investigated the prevalence and phenotype of TAMs in privately owned, treatment-naïve dogs with naturally occurring HSA. We used CD204 as a general macrophage marker and CD206 as a marker for M2-polarized macrophages. Formalin-fixed paraffin-embedded tissues from HSAs in the spleen (n = 9), heart (n = 6), and other locations (n = 12) from 17 dogs were sectioned and immunohistochemically labeled with CD204 and CD206 antibodies. The mean number of log(CD204)- and log(CD206)-positive cells and the ratio of log(CD206/CD204)-positive cells were compared with normal surrounding tissues and between tumor locations. There were significantly more macrophages and M2 macrophages, and a higher ratio of M2 macrophages to total macrophages in tumor hot spots (P = .0002, P < .0001, and P = .0002, respectively) and in tumor tissues outside of hot spots (P = .009, P = .002, and P = .007, respectively) than in normal surrounding tissues. There were no significant differences between tumor locations, but there was a trend toward higher numbers of CD204-positive macrophages within the splenic tumors. There was no association between histological parameters or clinical stage and TAM numbers or phenotype. As in humans, TAMs in dogs with HSA have a predominantly M2-skewed phenotype. Dogs with HSA could serve as excellent models to evaluate new TAM-reprogramming therapies.

The Distribution of IgT mRNA+ Cells in the Gut of the Atlantic Salmon (Salmo salar L.).

The newly discovered IgT+ B cell is thought to play a dominant role in mucosal immunity, but limited studies have examined its distribution in fish species, hindering our understanding of its function. This study investigated IgT and poly Ig receptor (pIgR) mRNA+ cell distribution in Atlantic salmon (Salmo salar) gut using RNAscope in situ hybridization (ISH) and assessed the effects of vaccination. The pyloric caeca, mid-intestine (first and second parts), and posterior segment in two weight stages (Group 1: avg. 153 g, Group 2: avg. 1717 g) were examined in both vaccinated and unvaccinated fish. ISH revealed more IgT mRNA+ cells in the second part of the midgut compared to other intestinal segments, as well as a higher number of positive cells in Group 2 (older fish). In line with previous findings, intraperitoneal vaccination had no significant impact on the number of IgT+ transcripts. IgT mRNA+ cells were found mostly in the lamina propria and near capillaries, while pIgR was registered in both the lamina propria and mucosa. Interestingly, vaccinated fish presented adhesions and granulomatous tissue in the peritoneum, with both IgT and pIgR mRNA+ cells. Taken together, these results suggest that the distribution of IgT mRNA+ cells in the intestine of Atlantic salmon is region-specific and is not affected by intraperitoneal vaccination but varies with fish age.

Red and melanized focal changes in white skeletal muscle in Atlantic salmon (Salmo salar): Comparative analysis of farmed, wild and hybrid fish reared under identical conditions.

Selective breeding plays a vital role in the production of farmed Atlantic salmon and has shown success in many aspects. Still, challenges related to fish health and welfare continue to result in significant economic losses. One such challenge is red and melanized focal changes (RFC/MFC), which result from acute and chronic inflammation, respectively, in the skeletal muscle. Importantly, RFC/MFC has not been observed in wild Atlantic salmon, suggesting that both external and genetic factors may contribute to the development of inflammation. To investigate the underlying cause of RFC/MFC, we conducted a study involving 1854 Atlantic salmon of farmed, wild and hybrid origin. All fish were reared under identical conditions to minimize the influence of external factors. Throughout the production cycle, the fish was monitored for growth parameters and examined for RFC/MFC using macroscopic and histological analysis. We found no association between the experimental groups and the presence of RFC/MFC. Histological investigations revealed melano-macrophages in the soft tissue in freshwater smolt, although no macroscopic discoloration was observed. MFC showed granulomas in various stages, suggesting a complex progression of the condition. In summary, we conclude that RFC/MFC is primarily caused by external factors found in the rearing facilities of farmed Atlantic salmon.

Anatomical and pathological characteristics of ribs in the Atlantic salmon (Salmo salar L.) and its relevance to soft tissue changes.

Studies on the anatomical and pathological characteristics of ribs in farmed Atlantic salmon (Salmo salar L.) are warranted due to their possible association with red and melanized focal changes (RFC and MFC) in the fillet, a major quality and animal welfare concern. In this work, we provide an anatomical description of ribs based on radiographical and histological analyses. We also address various pathological rib changes and their association to RFC and MFC. In total, 129 fish were investigated; captured wild (n = 10) and hatchery reared (n = 119) Atlantic salmon (3.5-6.1 kg). The fish were selected based on the macroscopic presence of RFC, MFC or no changes (controls). Radiographic results revealed costal abnormalities in all fish groups. By histological investigations of the variations herein, our results provide new insight into the anatomical characteristics including vascularization within the ribs; a potential site for haemorrhage following costal fractures. Costal fractures were detected by radiology in 40 of 129 samples (RFC: 38.4%, MFC: 47.2%, controls: 9.5 %). A statistically significant association was found between costal fractures and red (p = 0.007) and melanized changes (p = 0.000). However, red and melanized changes were also observed in samples with no costal fractures (n = 45), indicating that also other factors influence the development of RFC/MFC.

In-depth health surveillance and clinical nutrition in farmed Atlantic salmon: a strategic attempt to detect and mitigate an HSMI outbreak.

Fish health personnel have limited tools in combatting viral diseases such as heart and skeletal muscle inflammation (HSMI) in open net-pen farmed Atlantic salmon. In this study, we aimed to predict HSMI by intensified health monitoring and apply clinical nutrition to mitigate the condition. We followed a commercial cohort (G1) of Atlantic salmon that was PRV-1 naïve when transferred to a sea cage at a location where HSMI outbreaks commonly occur. The fish in the other cages (G2-G6) at the location had a different origin than G1 and were PRV-1 positive prior to sea transfer. By continuous analysis of production data and sequentially (approximately every fourth week) performing autopsy, RT-qPCR (for PRV-1 and selected immune genes), blood and histological analysis of 10 fish from G1 and G2, we identified the time of PRV-1 infection in G1 and predicted the onset of HSMI prior to any clinical signs of disease. Identical sequences across partial genomes of PRV-1 isolates from G1 and G2 suggest the likely transfer from infected cages to G1. The isolates were grouped into a genogroup known to be of high virulence. A commercial health diet was applied during the HSMI outbreak, and the fish had low mortality and an unaffected appetite. In conclusion, we show that fish health and welfare can benefit from in-depth health monitoring. We also discuss the potential health value of clinical nutrition as a mean to mitigate HSMI.

An Atypical Course of Cardiomyopathy Syndrome (CMS) in Farmed Atlantic Salmon (Salmo salar) Fed a Clinical Nutrition Diet.

Cardiomyopathy syndrome (CMS) poses a significant threat to farmed Atlantic salmon (Salmo salar), leading to high mortality rates during the seawater phase. Given that controlled experimental challenge trials with PMCV do not reproduce the mortality observed in severe field outbreaks of CMS, field trials on natural CMS outbreaks are warranted. This field study explored the impact of a clinical nutrition intervention, specifically a diet enriched with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), on a severe CMS outbreak in a commercial sea farm. CMS was diagnosed in a single sea cage with high mortality rates. Histopathological analysis, RT-qPCR in situ hybridization for virus detection, and fatty acid composition analysis were used to monitor the impact of disease and the inclusion of EPA and DHA in heart tissue. Following the implementation of clinical nutrition, a decline in mortality rates, regression of CMS-associated changes, and a significant reduction in piscine myocarditis virus (PMCV) RNA load were observed within the salmon population. Fatty acid composition analysis of heart samples demonstrated increased levels of EPA and DHA, reinforcing the association between dietary factors, viral load dynamics, and overall fish health. Although further validation is needed in future studies, as field trials may not be sufficient to establish causation, our results indicate that optimizing the EPA + DHA levels may prove beneficial in severe CMS outbreaks.

Early immunohistochemical detection of pulmonary micrometastases in dogs with osteosarcoma.

BACKGROUND: Despite decades of research, the early phases of metastatic development are still not fully understood. Canine osteosarcoma (OS) is a highly aggressive cancer, with a high metastatic rate (> 90%), despite a low overt metastatic prevalence at initial diagnosis (< 15%). Canine OS is generally regarded as a good clinically relevant model for human OS. The aim of this hypothesis-generating study was to evaluate a method to detect pulmonary micrometastases and study their prevalence in dogs with OS without macroscopic metastases. We prospectively enrolled dogs with OS that received no cancer-specific treatment (n = 12) and control dogs without cancer (n = 2). Dogs were necropsied and sampled immediately after euthanasia. The OS dogs were classified as having macroscopic metastases (n = 2) or not (n = 10). We immunohistochemically stained one tissue sample from each of the seven lung lobes from each dog with a monoclonal antibody (TP-3) to identify micrometastases (defined as clusters of 5-50 tumour cells), microscopic metastases (> 50 tumour cells) and TP-3 positive single cells (< 5 tumour cells). RESULTS: We showed that pulmonary micrometastases easily overseen on routine histology could be detected with TP-3. Pulmonary micrometastases and microscopic metastases were present in two dogs with OS without macroscopic metastases (20%). Micrometastases were visualised in three (43%) and four (57%) of seven samples from these two dogs, with a mean of 0.6 and 1.7 micrometastases per sample. Microscopic metastases were present in one (14%) and four (57%) of seven samples from the same two dogs, with a mean of 0.14 and 1.0 microscopic metastases per sample. There were four (57%) and two (29%) samples with neither microscopic metastases nor micrometastases for each of these two dogs. The prevalence of pulmonary micrometastases (20%) was significantly lower than expected (> 90%) based on commonly expected metastatic rates after amputation (P < 0.0001). There was no statistically significant difference in the number of TP-3 positive single cells in between groups (P = 0.85). CONCLUSIONS: Pulmonary micrometastases could be detected with TP-3 immunohistochemistry in a subset of dogs with OS before macroscopic metastases had developed. We propose that dogs with spontaneous OS represent clinically relevant models to study early micrometastatic disease.

PRV-1 Infected Macrophages in Melanized Focal Changes in White Muscle of Atlantic Salmon (Salmo salar) Correlates With a Pro-Inflammatory Environment.

Melanized focal changes in white skeletal muscle of farmed Atlantic salmon, "black spots", is a quality problem affecting on average 20% of slaughtered fish. The spots appear initially as "red spots" characterized by hemorrhages and acute inflammation and progress into black spots characterized by chronic inflammation and abundant pigmented cells. Piscine orthoreovirus 1 (PRV-1) was previously found to be associated with macrophages and melano-macrophages in red and black spots. Here we have addressed the inflammatory microenvironment of red and black spots by studying the polarization status of the macrophages and cell mediated immune responses in spots, in both PRV-1 infected and non-infected fish. Samples that had been collected at regular intervals through the seawater production phase in a commercial farm were analyzed by multiplex fluorescent in situ hybridization (FISH) and RT-qPCR methods. Detection of abundant inducible nitric oxide synthase (iNOS2) expressing M1-polarized macrophages in red spots demonstrated a pro-inflammatory microenvironment. There was an almost perfect co-localization with the iNOS2 expression and PRV-1 infection. Black spots, on the other side, had few iNOS2 expressing cells, but a relatively high number of arginase-2 expressing anti-inflammatory M2-polarized macrophages containing melanin. The numerous M2-polarized melano-macrophages in black spots indicate an ongoing healing phase. Co-localization of PRV-1 and cells expressing CD8+ and MHC-I suggests a targeted immune response taking place in the spots. Altogether, this study indicates that PRV-1 induces a pro-inflammatory environment that is important for the pathogenesis of the spots. We do not have indication that infection of PRV-1 is the initial causative agent of this condition.

Anatomy of teleost fish immune structures and organs.

The function of a tissue is determined by its construction and cellular composition. The action of different genes can thus only be understood properly when seen in the context of the environment in which they are expressed and function. We now experience a renaissance in morphological research in fish, not only because, surprisingly enough, large structures have remained un-described until recently, but also because improved methods for studying morphological characteristics in combination with expression analysis are at hand. In this review, we address anatomical features of teleost immune tissues. There are approximately 30,000 known teleost fish species and only a minor portion of them have been studied. We aim our review at the Atlantic salmon (Salmo salar) and other salmonids, but when applicable, we also present information from other species. Our focus is the anatomy of the kidney, thymus, spleen, the interbranchial lymphoid tissue (ILT), the newly discovered salmonid cloacal bursa and the naso-pharynx associated lymphoid tissue (NALT).

Variations in mucous cell numbers in gills of Atlantic salmon (Salmo salar) presmolt in commercial freshwater farms in Norway.

Fish gills are heavily exposed to the external milieu and may react against irritants with different cellular responses. We describe variations in mucous cell counts in gills from healthy Atlantic salmon (Salmo salar) presmolts in five recirculating aquaculture system (RAS) farms and one flow-through farm. Based on certain criteria, mucous cells were histologically quantified in a defined lamellar region of the gills and the counts were analysed. Immunohistochemistry (IHC) was used to investigate epithelial responses. The median number of total mucous cells in the defined region was 59 per fish. Between the farms, the medians varied from 31 to 101 with the lowest in the flow-through farm. A regression model was fitted with "total mucous cells" as the dependent variable and with "fish length" and "fish farm" as independent variables. The proportion of variation in mucous cell counts explained by the model was twice as high when "fish farm" was included compared to only "fish length." IHC revealed proliferative responses in coherence with high mucous cell numbers. Conclusively, the variation in mucous cell counts depends on combined farm-related factors. Establishing a baseline for mucous cell counts is fundamental in the development of high-throughput monitoring programmes of gill health in farmed fish.

IgM+ and IgT+ B Cell Traffic to the Heart during SAV Infection in Atlantic Salmon.

B cells of teleost fish differentiate in the head kidney, and spleen, and either remain in the lymphatic organs or move to the blood and peripheral tissues. There is limited knowledge about piscine B cell traffic to sites of vaccination and infection and their functional roles at these sites. In this work, we examined the traffic of B cells in Atlantic salmon challenged with salmonid alphavirus (SAV). In situ hybridization (RNAScope) showed increased numbers of immunoglobin (Ig)M+ and IgT+ B cells in the heart in response to SAV challenge, with IgM+ B cells being most abundant. An increase in IgT+ B cells was also evident, indicating a role of IgT+ B cells in nonmucosal tissues and systemic viral infections. After infection, B cells were mainly found in the stratum spongiosum of the cardiac ventricle, colocalizing with virus-infected myocardial-like cells. From sequencing the variable region of IgM in the main target organ (heart) and comparing it with a major lymphatic organ (the spleen), co-occurrence in antibody repertoires indicated a transfer of B cells from the spleen to the heart, as well as earlier recruitment of B cells to the heart in vaccinated fish compared to those that were unvaccinated. Transcriptome analyses performed at 21 days post-challenge suggested higher expression of multiple mediators of inflammation and lymphocyte-specific genes in unvaccinated compared to vaccinated fish, in parallel with a massive suppression of genes involved in heart contraction, metabolism, and development of tissue. The adaptive responses to SAV in vaccinated salmon appeared to alleviate the disease. Altogether, these results suggest that migration of B cells from lymphatic organs to sites of infection is an important part of the adaptive immune response of Atlantic salmon to SAV.

Anatomy, immunology, digestive physiology and microbiota of the salmonid intestine: Knowns and unknowns under the impact of an expanding industrialized production.

Increased industrialized production of salmonids challenges aspects concerning available feed resources and animal welfare. The immune system plays a key component in this respect. Novel feed ingredients may trigger unwarranted immune responses again affecting the well-being of the fish. Here we review our current knowledge concerning salmon intestinal anatomy, immunity, digestive physiology and microbiota in the context of industrialized feeding regimes. We point out knowledge gaps and indicate promising novel technologies to improve salmonid intestinal health.

Lymphoid Tissue in Teleost Gills: Variations on a Theme.

In bony fish, the gill filaments are essential for gas exchanges, but also are vulnerable to infection by water-borne microorganisms. Omnipresent across fish, gill-associated lymphoid tissues (GIALT) regulate interactions with local microbiota and halt infection by pathogens. A special GIALT structure has recently been found in Salmonids, the interbranchial lymphoid tissue (ILT). However, the structural variation of GIALT across bony fish remains largely unknown. Here, we show how this critical zone of interaction evolved across fishes. By labeling a conserved T-cell epitope on tissue sections, we find that several basal groups of teleosts possess typical ILT, while modern teleosts have lymphoepithelium of different shape and size at the base of primary gill filaments. Within Cypriniformes, neither body size variation between two related species, zebrafish and common carp, nor morphotype variation, did have a drastic effect on the structure of ILT. Thereby this study is the first to describe the presence of ILT in zebrafish. The ILT variability across fish orders seems to represent different evolutionary solutions to balancing trade-offs between multiple adaptations of jaws and pharyngeal region, and immune responses. Our data point to a wide structural variation in gill immunity between basal groups and modern teleosts.

Immunopathological characterization of red focal changes in Atlantic salmon (Salmo salar) white muscle.

Farmed Atlantic salmon (Salmo salar) are prone to various conditions affecting the quality of the fillet. A well-known but so far poorly understood condition is the focal red changes in muscle, often referred to as haemorrhages. Such changes are characterized by muscle necrosis, haemorrhages and acute inflammation. They can progress into focal melanised changes, a chronic inflammatory condition with melanin-producing leukocytes. The initial cause of intramuscular haemorrhages is unknown. In this study, we aimed to reveal some of their key immunological features. Samples of red focal changes were investigated by immunohistochemistry (IHC), in situ hybridization (ISH) and RT-qPCR for various immune markers. The results were compared with samples of melanised changes and control muscle, subjected to the same analyses. In all red changes, infiltrates with mononuclear cells were detected, consisting mostly of MHC class I/II+ cells, but also of CD3+ and CD8+ cells. ISH studies on IgM showed few to moderate amounts of B-cells in red focal changes. Trends in the RT-qPCR showed upregulation of genes related to innate immunity in the red changes, whereas genes related to adaptive immunity were upregulated in the melanised changes. An important result was the significant downregulation of the anti-inflammatory cytokine IL10 in all red changes. Our findings indicate that we can rule out an auto invasive nature of the changes. The downregulation of IL10 at an early phase is a trait for the condition.