To our knowledge, this study is the first one to describe the muscle fiber ultrastructure of IIM patients in clinical phenotypes ADM, DM, JDM, PM plus anti-synthetase syndrome (ASS) and scleromyositis by SEM. Since the unexplore application of SEM in these patients and the lack of classification criteria including SEM findings, we decided to categorize and describe the muscle fiber ultrastructure according to muscle damage and muscle weakness markers represented by muscle enzymes and MMT8 score, respectively. To complement the obtained information on muscle fiber ultrastructure, we also characterized the immunological profile of IIM patients aiming to get a better understanding of their physiopathology.
Serum levels of muscle enzymes are considered a support tool for myositis diagnostic considered in EULAR/ACR 2017 criteria for IIM patient classification. Is important to remind that serum levels of muscle enzymes might reflect the muscle damage or the lack of physical training in some IIM patients 10 meanwhile, the MMT8 score is a validated tool for muscle weakness evaluation in myositis patients11, therefore both parameters are considered adequate markers for muscle damage and muscle weakness.
According to muscle fiber ultrastructure description, we presented the SEM images in decreasing order of the MMT8 score. Only two of ten patients had a MMT8 maximum score, the muscle tissue of these patients included muscle fibers with lineal morphology, similar size and were disposed of in fascicle conformation. However, while muscle strength decreased, we observed more irregularities, porosities, abundant leukocyte infiltration as well as disruption of linearity and fascicle conformation, even in the patients with the lowest MMT8 score, we observed muscle fiber destruction with multiple perforations and difficulty to identify muscle fiber and muscle fascicle structures. Likewise, muscle enzymes elevation became evident as muscle strength decreases, this is in concordance with the observed correlation between serum levels of muscle enzymes and the MMT8 score.
An important aspect to consider regarding the muscle fiber description of our patients is the fact of the observed discordances between optical and electronic microscopy; according to HE staining, we did not observe any finding suggestive of muscle pathology in three of our patients, meanwhile, we found alterations in the ultrastructure of muscle fiber of the same patients. This situation denotes the usefulness of SEM as a tool with higher resolution to get a better opportunity to observe histological changes and a better comprehension of the muscle tissue alterations of IIM patients.
Other important fact regarding muscle histological examination of IIM patients is the discordance between clinical and histopathological diagnosis that has been previous reported indicating up to 55% of change in diagnosis after muscle biopsy in IIM patients. Muscle biopsy examination has also a great utility in deep characterization of IIM patients12. In our study, the clinical phenotype was not concordant in 30% with muscle biopsy results; this is one of the reasons why the decision of muscle biopsy is discussing nowadays because the lack of expertise in its interpretation. These findings highlight the importance of muscle biopsy for an accurate diagnosis and classification of IIM patients.
In addition to muscle fiber description, we also aimed to characterize the immunological profile of our study group by serum detection of MSA and MAA as well as cytokines and chemokines quantification. We found a seropositivity of 41.7% of MSA or MAA in concordance with the phenotypes previously described as the case of the presence of anti-MDA5 in ADM phenotype or ILD development in anti-EJ seropositive patients13,14.
Regarding cytokines, it is important to denote that not all cytokines or chemokines were detected because their serum levels were lower than the minimum considered in the detection range, we considered them as zero for comparisons and reported the detection rate in Table 3. In this context, cytokines such as IFN-α2, IL-6, IL-23, and chemokine CCL2 had a detection rate of 100% meanwhile IL-12p70 and TNF-α were not detected in the serum of IIM patients and the cytokine IL-17 was detected in fewer patients probably due to immunosuppressive therapy.
We found higher levels of IL-6 in IIM patients in respect to control group. This cytokine is one of the main mediators of pro-inflammatory responses, mainly secreted by macrophages, fibroblasts and endothelial cells15; IL-6 promotes vascular endothelial activation by an increase of E-selectin, intercellular adhesion molecule 1 (ICAM-1) and vascular cellular adhesion molecule 1 (VCAM-1) expression which is important for leukocyte migration to muscle tissue16, otherwise, IL-6 elevated levels have been reported in other rheumatic diseases including rheumatoid arthritis, Sjögren’s syndrome, and Crohn’s disease17–19. The role of IL-6 in IIM has been previously reported as the case of the model of myosin-induced experimental myositis where mice developed myopathy, but IL-6 deficient mice did not show clinical nor histological signs of muscle damage20. Additionally, elevated serum levels of IL-6 in IIM patients have been also reported and proposed as an activity disease biomarker in DMJ21. Considering this background as well as our results, we could consider IL-6 as a one of the main mediators of tissue destruction in IIM immunopathology.
Otherwise, IFNg and IL-17A which are related cytokines to IIM pathogeny because of their participation in MHC-I overexpression and autoimmunity development22,23 did not show difference. This behavior might be explained by the clinical remission of the IIM, the immunosuppressive treatment or their low concentrations of cytokines and chemokines in peripheral blood, however, it would be important to determine the expression of these molecules directly in muscle tissue.
Afterwards, according to MSA/MAA seropositivity we observed only a tendency to IL-6 higher levels in seropositive patients. On the other hand, we found higher levels of IFN-γ and CCL2 in seropositive patients. To our knowledge, this is the first report comparing cytokine and chemokine serum levels according MSA/MAA seropositivity, however, due to small sample size, we could not carry out the analysis for each autoantibody. We propose the comparative of cytokine and chemokine serum levels according to each MSA and MAA as a perspective for a better understanding of the immunological profile of IIM patients.
The implication of IFNs in IIM is well-known and has been widely reported since the first report in 198624. This cytokine is mainly produced by monocytes, macrophages, and natural killer 25 cells after pattern recognition receptors (PRR) activation, but IFN-γ is also synthesized by TCD4+ lymphocytes (Th1 profile) and TCD8+ lymphocytes26. It has been demonstrated that the IFN-γ gene is overexpressed in myofibers surrounded by TCD8+ lymphocytes27. Otherwise, it is highly recognized that this cytokine induces the MHC-I and MHC-II overexpression in human myoblasts28,29 which is associated with IIM immunopathology because of the facilitation of myofibers recognition by TCD8+ lymphocytes16 as well as the trigger of the stress of endoplasmic reticulum30.
On the other hand, it has been reported that the chemokine CCL2, mainly produced by monocytes and macrophages, participates in IIM pathogenesis by the stimulation of leukocyte migration to the muscle tissue31. In addition, the inflammatory infiltrates which surround the blood vessels of muscle tissue are mainly composed of lymphocytes and macrophages that are target cells of CCL216. It has been shown distinct evidence of CCL2 implication in myositis including its expression at mRNA level in patient muscle tissue, CCL2 protein expression in endothelial cells of IIM patients as well as higher levels of CCL2 in muscle tissue of IIM patients32,33. Moreover, we could infer that CCL2 synthesis could be induced by membrane attack complex 25 formation in endothelial cells surface of DM patients, the main phenotype in our study. Otherwise, there are few reports which suggest the consideration of this cytokine as an early biomarker for ILD development since CCL2 elevated levels have been observed in DM/PM patients with ILD complication34,35. However, in the present study, it was not possible to associate cytokine and chemokine serum levels with these complications.
Once we characterized the damage and muscle strength as well as immunological profile, we looked for correlations between these parameters. We did not find any association according to the MMT8 score, however, one of the most remarkable findings is the association of LDH enzyme with immunological profile reflected in the correlation between LDH and IFN-γ serum levels, the difference of IFN-γ serum levels according to LDH normal or elevated levels, higher LDH levels in seropositive patients, as well as a tendency to the higher frequency of seropositive patients with LDH, elevated levels.
Concerning a specific histopathological finding and its association with immunological profile, we observed that the most common finding according to HE results, was perivascular inflammatory infiltrate. The patients with this feature had higher levels of ESR which reflects the pro-inflammatory status and activation of leukocyte migration. We also observed a decreased MMT8 score and increased of muscle enzymes when there was presence of regeneration markers (basophilic fibers) as well as lower muscle strength and IL-18 elevated serum levels when the patients had endomysial inflammatory infiltrate.
This infiltrate in muscle tissue is commonly composed by CD8+ T cells, which could have an association with this cytokine since it has been proved that IL-18 receptor (IL-18R) has higher expression in functional phenotypes of CD8+ T cells36 meanwhile other studies have demonstrated that exhausted CD8+ T cells downregulates its IL-18R expression37. In addition, IL-18 has been proposed as a biomarker in IIM due to its correlation with activity disease38, and its increment in serum as consequence of muscle damage39. Despite the low number of patients in our study, we were able to find an association of higher IL-18 levels in patients with endomysial infiltrate probably due to an active state of immune response mainly orchestrated by CD8+ T cells.
Moreover, the most common findings by SEM included myofiber surface irregularities, altered muscle morphology and non-linear muscle fibers. We observed lower levels of IL-1β and CXCL8 in patients with altered muscle morphology, probably due to a chronic process.
We looked for a possible association between HE and SEM findings (Supplementary Table 3), we observed a tendency of basophilic fibers when they were lineal without cellular infiltrate, which could suggest that patients with these features are probably in clinical remission.
Regarding immunosuppressive treatment, we find lower levels of CXCL8 in patients treated with prednisone. We also found a tendency to diminished levels of IL-23 in patients treated with mofetil mycophenolate furthermore, we could not detect IL-1β neither IL-17A, important cytokines for IIM immunopathogeny23, in the serum of these patients.
Prednisone is a synthetic glucocorticoid biologically inert and converted to prednisolone in the liver, its immunosuppressant activity is because of the inhibition of prostaglandin and leukotriene synthesis, these molecules are implicated in vascular and cellular processes of inflammation which reduces vasodilatation, capillary permeability and the leukocyte migration40. Regarding CXCL8, is a chemokine secreted by endothelial cells in the inflammation site which possess a role in leukocyte recruitment and transmigration41, moreover, if the prednisone reduces the vasodilatation and capillary permeability, we could infer there are no stimulation of the endothelial cells and the CXCL8 serum concentration decreases in these patients.
Concerning mofetil mycophenolate, an inhibitor of the inosine-5'-monophosphate dehydrogenase, an enzyme responsible for de novo synthesis of guanine nucleotides, an important process for the proliferation of T and B lymphocytes41. Is the treatment with the highest quantity of diminished serum levels of cytokines (Supplementary Table 1), probably because of the direct inhibition of immune cell proliferation which is reflected in a lower autoantibody production as well as lower cytokine and chemokine production. There are no previous reports regarding the direct effect of mycophenolate in IL-23, IL-1β and IL-17A synthesis, however, these three cytokines are involved in an axis that amplifies the proliferation of Th17 cells which produce IL-17A, a cytokine that in turn, stimulates the IL-1, IL-6 and TNF-α production42, moreover, if mofetil mycophenolate diminishes the leukocyte proliferation, these are not able to differentiate, thus the IL-23, IL-1 and IL-17 axis is interrupted, thus, it could explain why IL-23 levels are diminished and IL-17 and IL-1β levels were not detected in our patients under mofetil mycophenolate treatment. It is important to denote that the implication of IL-17 and IL-23 in IIM has been previously reported as an increment of both cytokines in the supernatant of the ex vivo culture of peripheral blood mononuclear cells of IIM patients with a recent disease establishment when were compared with patients with established disease43. Likewise, it has been reported an increment of IL-23 serum levels in IIM patients as well as a higher expression of the cytokine in impaired muscle tissue, even IL-23 has been proposed as a therapeutic target for IIM treatment44.
When we analyzed if there were difference in cytokine and chemokine serum levels according to the quantity of immunosuppressive drugs prescript because of therapy, we observed that IL-18 remains elevated even in patients treated with three distinct immunosuppressive treatments, thus we can highlight the importance of IL-18 in the disease activity as well as the fact that there are cytokines that even under immunosuppressive treatment cannot be diminished45.
In summary, we were able to find and describe muscle fiber ultrastructure with marked irregularities, porosities, disruption in the linearity and integrity of the fascicle, more evident in patients with increased serum levels of muscle enzymes and diminished muscle strength. Likewise, there was a negative correlation between the increment of muscle enzymes and the MMT8 score.
We did not observe a particular association of clinical phenotype with histopathological nor serological findings, moreover, is important to consider the clinical stage of the IIIM (most of them in clinical remission). Concerning the association between immunological parameters, the serum levels of IFN-γ are higher in MSA/MAA seropositive patients, regarding to muscle and immunological parameters association, the LDH enzyme is the muscle damage marker even unspecific of muscle damage with a higher association to immunological profile.
In conclusion, even the scarce reports about the use of SEM as a tool in all clinical phenotypes of IIM, our work provides an excellent opportunity to discuss and reframe the clinical usefulness of SEM in the diagnostic approach of IIM.