Apoptosis in idiopathic inflammatory myopathies with partial invasion; a role for CD8+ cytotoxic T cells?

Olof Danielsson; Bo Häggqvist; Liv Gröntoft; Karin Öllinger; Jan Ernerudh

doi:10.1371/journal.pone.0239176

. 2020 Sep 16;15(9):e0239176. doi: 10.1371/journal.pone.0239176

Apoptosis in idiopathic inflammatory myopathies with partial invasion; a role for CD8⁺ cytotoxic T cells?

Olof Danielsson ^1,^*, Bo Häggqvist ¹, Liv Gröntoft ¹, Karin Öllinger ², Jan Ernerudh ³

Editor: Alfred S Lewin⁴

PMCID: PMC7494097 PMID: 32936839

Abstract

Polymyositis and inclusion body myositis are idiopathic inflammatory myopathies, with a pathology characterized by partial invasion of non-necrotic muscle fibres by CD8⁺ cytotoxic T-cells, leading to fibre degeneration. Although the main effector pathway of CD8⁺ T-cells is to induce apoptosis of target cells, it has remained unclear if apoptosis occurs in these diseases, and if so, if it is mediated by CD8⁺ T-cells. In consecutive biopsy sections from 10 patients with partial invasion, muscle fibres and inflammatory cells were assessed by immunohistochemistry and apoptotic nuclei by the TUNEL assay. Analysis of muscle fibre morphology, staining pattern and quantification were performed on digital images, and they were compared with biopsies from 10 dermatomyositis patients and 10 controls without muscle disease. Apoptotic myonuclei were found in muscle with partial invasion, but not in the invaded fibres. Fibres with TUNEL positive nuclei were surrounded by CD8⁺ T-cells, granzyme B⁺ cells and macrophages, but lacked FAS receptor expression. In contrast, apoptotic myonuclei were rare in dermatomyositis and absent in controls. The findings confirm that apoptosis occurs in idiopathic inflammatory myopathies and support that it is mediated by CD8⁺ cytotoxic T- cells, acting in parallel to the process of partial invasion.

Introduction

Idiopathic inflammatory myopathies (IIM) constitute a heterogeneous group of diseases with a presumed autoimmune pathogenesis [1]. They are commonly classified on the basis of clinicopathologic features [2], although variations due to overlapping or unspecific clinical and pathological findings sometimes make diagnosis and classification a challenge [3–5]. Key pathologic findings, distinguishing the inflammatory myopathies, are the localization of the inflammatory infiltrates and the different types of muscle fibre degeneration [5, 6]. Muscle biopsies from patients with polymyositis (PM) and inclusion body myositis (IBM) characteristically show invasions of major histocompatibility complex class I—(MHC I) expressing muscle fibres by an inflammatory infiltrate, dominated by CD8⁺ cytotoxic T cells [6]. This scenario is commonly referred to as a partial invasion, and is included as a classification criterion for PM, intended for research studies [7]. The invaded fibre does not show classical signs of necrosis, but rather a gradual disintegration and displacement by inflammatory cells [8]. This type of cell degeneration is not found in other tissues, and is rarely seen in other muscle diseases [6]. In dermatomyositis (DM), considered as a humorally mediated microangiopathy affecting muscle and skin, the most characteristic pathologic muscle findings are perivascular inflammatory infiltrates and perifascicular atrophy [2]. Another widely accepted pathology based subgroup of IIM is immune-mediated necrotizing myopathy (IMNM), also called necrotizing autoimmune myopathy (NAM) [7], characterized by muscle fibre necrosis and the relative absence of inflammatory cells. In addition, there is an ongoing discussion about further myopathological subdivisions of these diseases [5, 9]. However, partial invasion is a feature that is common to both IBM and PM (strictly defined), and extensive sectioning of muscle tissue shows that partial invasion is sometimes encountered also in overlap syndromes, and, rarely, in classical DM [4, 6]. This raises the question whether there are further shared pathogenic processes, associated with partial invasion.

The general effector mechanism mediated by cytotoxic CD8⁺ T-cells is to induce apoptosis, either by means of granzyme/perforin secretion or by binding of the FAS-ligand (FAS-L) to FAS receptors (hereafter called FAS) of target cells [10–13]. Expression of granzyme B and perforin in inflammatory cells [14, 15], as well as the expression of FAS in muscle fibres [16–18], has been reported in inflammatory myopathies, but signs of apoptotic fibre nuclei have rarely been observed in fibres affected by partial invasion [19, 20]. Overall, signs of apoptosis in IIM muscle fibres have, despite reports of its presence [20, 21], been considered rare, and unlikely of importance for the pathogenesis [20, 22]. Absence of apoptosis has also been reported for the inflammatory cells in IIM, which has been interpreted as an inability to efficiently terminate the inflammatory reaction, possibly contributing to the chronicity of these diseases [22, 23].

The pathogenic role of CD68⁺ macrophages, the second most frequent cell type in the invading infiltrate [6], is still unclear. Notably, no ultrastructural signs of phagocytosis have been noted [8], and the macrophages express markers of myeloid dendritic cells [24], stressing their antigen presenting potential. Macrophages within the invading infiltrate have further been suggested to contribute to the distinctive pathology by producing inflammatory molecules [25]. Macrophages expressing the scavenger receptor CD163 (type 2 macrophages) have been attributed anti-inflammatory properties, mediated by IL-10 [26], and a regenerating role in muscle [27]. On the other hand, a correlation between disease activity in IIM muscle and CD163⁺ macrophages has been reported [28, 29], but their relative number in relation to CD68⁺ macrophages has not been investigated.

Considering the main cytotoxic effector functions of the CD8⁺ T-cells, characteristically present in IBM and polymyositis, several studies have identified factors with potential to protect muscle from apoptosis [16, 18, 30, 31]. The fact that muscle fibres do not normally express MHC I [32], but constitutionally HLA-G [33], and the rather uncommon occurrence of inflammatory muscle diseases, indicate a relative protected immunologic status of striated muscle. In line with this, we earlier reported a constitutional expression of Bcl-2, a major anti-apoptotic protein, in muscle from healthy controls, but lower Bcl-2 expression in PM and Duchenne muscular dystrophy [18]. Bcl-2 has potential to protect against granzyme-mediated apoptosis [34], but its precise role in muscle is unknown. Heat shock proteins (HSP) have cytoprotective functions that may be of importance [35], considering that the sarcoplasmatic reticulum stress, observed in IIM, may lead to apoptosis [36]. HSP70 has recently been shown to protect against apoptosis in cardiomyocytes [37]. Moreover, HSP70 has been detected in inflammatory myopathies [38, 39] and, also in this scenario, been attributed a protective function [40].

The aim of the study was to investigate if apoptotic myonuclei are present in muscle in the specific subgroup of IIM with partial invasion, and to detect the presence of key molecules of the apoptotic-mediating or -protective pathways in the affected and in adjacent fibres. The CD8⁺ T-cell-mediated immune effector mechanism and frequency of type 2 macrophages were evaluated.

Methods

Patients and biopsies

All biopsies were taken from the anterior tibial muscle for diagnostic purposes. We included stored tissue samples, obtained 1997–2002, with confirmed partial invasion from 10 patients (median age 62 years, range 32–75) with inflammatory myopathies (Table 1), which in a previous study [4] had been classified according to the Amato/European neuromuscular centre (ENMC) classification [7]. These samples were compared with two control groups, comprising 10 patients in each group. As an IIM control group, biopsies from patients with DM were selected; the reason for this choice was that this disease has another type of, more or less pathognomonic, pathological findings [7]. The DM cases (median age 52 years, range17-78) were consecutive biopsies investigated in our lab, where the available clinical and laboratory data allowed the classification of at least probable DM, according to the Amato/ENMC. Eight cases had a definite and two a probable DM according to this classification, and all cases had a definite DM according to the Bohan and Peter. The other control group comprised consecutive cases (median age 46 years, range 39–59), where neuromuscular disease had been excluded, based on biopsy and clinical examination. The cases in these latter two groups were included during April 2015. For demographic details of all cases, see S1 Dataset.

Table 1. Demography, diagnosis^* and results^ (number of partial invasions, TUNEL stained myonuclei, and necrotic fibres) of patients with partial invasion^*.

Case	Age (yrs)	Sex	Bohan & Peter	Amato/ENMC	Partial inv.	Necrotic fibres	TUNEL+ fibres	TUNEL+ infl. cells
1	66	m	IBM def.	IBM def.	1	0	1	0
2	62	f	Overlap IIM pr.	non IIM#	3	1	0	1
3	58	f	Overlap IIM def.	Poss. DM s. D.	4	0	1	0
4	32	f	PM def.	PM def.	1	1	0	1
5	66	f	Overlap IIM pr.	PM def.	3	0	1	7
6	53	f	PM poss.	non IIM#	1	0	0	1
7	60	m	IBM def.	IBM def.	1	2	0	3
8	73	m	IBM poss.	IBM poss.	3	1	0	2
9	70	m	IBM def.	IBM def.	1	0	2	3
10	75	f	PM pr.	PM def.	2	0	3	3

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*The diagnoses were according to the classifications of Bohan & Peter[41] and Amato/ European Neuromuscular Centre (ENMC) [7]. The IBM diagnosis was in both classifications according to Griggs [42].

**The investigated muscle section area was 1.37 mm².

*** The creatine kinase was elevated in all patients. # These two patients did not have a detectable muscle weakness and could thus not be classified according to Amato/ENMC. m: male, f: female, def.: definitive, pr.: probable, poss.: possible, IIM: idiopathic inflammatory myopathy, non-IIM: patients without weakness does not qualify as IIM according to Amato/ENMC, IBM: inclusion body myositis, PM: polymyositis, poss. DM s. D: possible dermatomyositis sine dermatitis.

Sectioning method and selection of cases with partial invasion

In the earlier study [4], muscle biopsies from 36 patients were shown to contain at least one partial invasion. In order to detect and verify strict biopsy criteria stated by Amato/ENMC [7], 35 consecutive cryosections had been made from each biopsy, and every 7^th section (6 μm) stained with haematoxylin eosin (HE). Partial invasions were first identified by the morphologic appearance in the HE stained sections and then confirmed by the following three sections, stained with antibodies against MHC I, CD8 and membrane attack complex (MAC). MAC was included to add sensitivity to detect myofibre necrosis [43]. For the present study, the frozen consecutive sections (-70°C) of the biopsies were thawed, and the HE stained sections were again studied in a Zeiss AXIO Imager light microscope. From the 36 cases, we selected those where the partial invasion were present in at least 3 HE sections (n = 10), and used the intermediate sections for immunohistochemistry and the TUNEL-assay.

Reflections on the representativeness of the included cases

The requirement that the partial invasion had to be detectable in 3 HE stained sections, had the effect that partial invasions with a smaller size than approximately 150 μm were not included. We have not found any report voicing the possibility that partial invasions of different sizes may differ in a qualitative manner, but it cannot be excluded. We earlier showed that partial invasions is present in many types of IIM [4]. However, it is more common and more frequent in IBM, followed by PM [44]. The distribution of diagnoses of investigated pi-cases is comparable with that found in the 36 cases in our 6 year IIM cohort [4] (Table 1). With the above reservation of pi-size, the results are thus considered representative for IIM with pi, not of any defined disease subgroup.

Immunohistochemistry

The sections were labelled with the following monoclonal antibodies: Bcl-2, CD68, CD163, FAS, Granzyme B, HSP70, Merosin 80, MyHC-fast, MyHC-slow and Spectrin (for details see Table 2). Primary antibodies were diluted in Da Vinci Green diluent (BiocareMedical, CA, USA) and incubated at room temperature in a humid chamber. Tris-buffered saline (TBS; 50 mM), pH 7.4, was used as washing buffer. Bound antibodies were detected using the Novolink Polymer Detection System (Leica Biosystems Nussloch GmbH, Germany), according to the manufacturer’s protocol. Novocastra DAB enhancer was used to improve the Bcl-2 visualization. Negative controls were performed by replacing the primary monoclonal antibody with IgG1 isotype control (X0931, Dako Denmark A/S) or by omitting the primary antibody.

Table 2. Antibodies and immunohistochemical procedures.

Antigen	Clone^¤	Manufacturer	Isotype	Incubation	Dilution	Fixation and time
Bcl-2	100	Santa Cruz^*	IgG1	18 h	1:10	Acetone, 10 min
CD8	C8/144B	DAKO^**	IgG1	30 min	1:200	PFA 2%, 8 min
CD68	KP1	Santa Cruz	IgG1	30 min	1:400	PFA 2%, 8 min
CD163	GHI/61	Santa Cruz	IgG1	30 min	1:400	PFA 2%, 8 min
FAS	B-10	Santa Cruz	IgG1	18 h	1:800	Acetone, 10 min
Granzyme B	GB11	Santa Cruz	IgG1	30 min	1:1600	PFA 2%, 8 min
HSP70	C92F3A-5	Santa Cruz	IgG1	30 min	1:400	PFA 2%, 8 min
Merosin 80	MAB1922	Millipore#	IgG1	30 min	1:100	Unfixed
MyHC-fast	WB-MHCf	Leica##	IgG1	30 min	1:200	Acetone, 10 min
MyHC-slow	WB-MHCs	Leica	IgG1	30 min	1:400	Acetone, 10 min
Spectrin	RBC2/3D5	Leica	IgG2b	30 min	1:50	PFA 2%, 8 min

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¤ All monoclonal antibodies were from mice and all incubations were at room temperature.

MyHC, myosin heavy chain; PFA: paraformaldehyde.

* Santa Cruz Biotechnology, Inc. Dallas TX;

** Dako Denmark A/S; # Millipore Corp, Temecula, CA; ## Leica Biosystems, Nussloch GmbH.

Detection of apoptotic nuclei

ApopTag® Plus Peroxidase In Situ Apoptosis Detection kit (EMD Milipore Corp., Temecula CA, USA) was used for TUNEL assay, according to the manufacturer’s protocol [45]. Of three consecutive sections, the first and third were stained, using the second as negative control. In addition to application of nuclease on biopsy samples and apoptosis-positive female rodent mammary glands, supplied by the manufacturer, human heart tissue from autopsy were used as positive controls. The heart tissue, friendly supplied by Erik Edston, had been used in an earlier study to detect apoptotic cardiomyonuclei [46], where the same TUNEL method was applied as in our study.

Complementary stains with spectrin, CD8 and granzyme B

In order to clearly discriminate, between apoptosis of myonuclei and immune cells, the sarcolemma of TUNEL stained sections was, after the first imaging session, re-stained with spectrin. The coverslips were removed and immunostaining was carried out according to manufacturer’s protocol, omitting the neutralization of endogenous peroxidase, as this had been carried out previously. In addition, in 3 cases no adherent granzyme B⁺ or CD8⁺ cells were detected in proximity of fibres with apoptotic myonuclei in the first staining session, and therefore the remaining adjacent sections were stained again with these antibodies.

Digital imaging and quantification

A digital image, using an AxioCam MRc-digital camera and Zen software (Carl Zeiss Microimaging GmbH, Jena, Germany), was obtained of each section, harbouring the earlier identified partial invasion. In the patients with DM, an area was identified, showing characteristic myopathic findings, such as a perifascicular atrophy (3 cases), other forms of perifascicular pathology (4 cases) or an inflammatory infiltrate (9 cases). A random area was chosen in the control biopsies. In each of the consecutive sections of the biopsies, the corresponding centre point was identified in the image. Using the Zen software, a circle with an area of 1.37 mm² around this centre point was drawn, and the stained cells and fibres in this area were counted. Spectrin stain was used to demarcate fibres in order to facilitate identification and counting, and the number of stained fibres and inflammatory cells was presented as a ratio per 100 fibres.

Statistical analyses

Statistical analysis was performed using the Prism program (Graphpad Software). The majority of data were not normally distributed; therefore, the Kruskal-Wallis test was used, followed by Dunn’s multiple comparison test. The Pearson normality test, however, confirmed normal distribution of the CD163/ CD68 ratio, hence the parametric ANOVA was used, followed by Tukey’s multiple comparisons test. P-values < 0.05 were considered significant and a prerequisite for performing the post-test.

Ethical approval

The biopsies had been taken from patients for diagnostic purposes. Written informed consent was obtained for using remaining tissue for research. The study was performed in accordance with relevant guidelines and regulations, and was approved by the regional Ethical Board in Linköping (ref. no. M58-07).

Results

IIM is associated with alteration in the expression of Bcl-2 and of the CD163/CD68 ratio

To provide an overview of apoptosis/anti-apoptosis and macrophage markers in the pathological processes, we first analysed the staining pattern in a circular area (1.37 mm²) surrounding partial invasions, and in areas (of the same size) containing an inflammatory infiltrate or a perifascicular pathology, in patients with DM. The selected areas of the partial invasion cases (pi-cases) contained 93–378 fibres (median 225), and of the DM-patients 132–414 (median 250) fibres. The findings of the IIM groups were also compared with the stains in a random area of the same size (containing 192–252 fibres (median 232)) in control biopsies, from patients without muscle disease.

There was a lower proportion of fibres expressing Bcl-2 in the two groups with inflammatory myopathies compared to controls (Fig 1A). The proportion of fibres expressing FAS, as well as fibres expressing HSP70, was higher in the pi-patients than in the controls, while the difference between DM-patients and the controls was not statistically significant (Fig 1B and 1C). Both the absolute numbers of CD68⁺ and CD163⁺ cells, and the ratio of CD163⁺ and CD68⁺ cells, were higher in the groups with inflammatory myopathies, compared to controls (Fig 1D–1F). Furthermore, the staining pattern of the CD68⁺ and the CD163⁺ cells indicated an expanded cytoplasm compatible with a dendritic morphology, which was not seen in the non-inflammatory controls cases (S1 Fig). In 4 out of 10 pi-cases, 5 necrotic fibres were found, and in 5 out of 10 DM cases, 14 necrotic fibres, whereas no necrotic fibre was detected in the controls.

Characteristics of partial invasion

In the selected area of the pi-cases (n = 10), 20 partial invasions were detected. The inflammatory cells were in all cases surrounded by a semi-opaque substance, tentative of a common matrix (Fig 2). Most infiltrates (18/20) showed clear signs of invasion also of a neighbouring fibre, and in a majority of the cases (13/20), the part of the infiltrate, directly interacting with the fibre, had a broad base (Fig 2A and 2B), although other morphological appearances of infiltrate-fibre interaction were also noted (Fig 2C and 2D).

Fig 2 — Four cases of partial invasion stained with haematoxylin-eosin (HE) are shown, where the inflammatory infiltrate is surrounded by a semi-opaque material (arrows). In the upper images (a,b) the infiltrate-fibre interaction is broad based and invasion of neighbouring fibres is seen. In the lower left image (c), the infiltrate is (in several consecutive sections) encapsulated within the fibre. The lower right image (d) shows the only case where inflammatory cells indented the fibre at multiple sites.

The spectrin stain, used in the diagnostic routine to detect membrane damage [47], showed undisrupted expression of the fibre membrane in 14 cases, while the membrane had unstained segments in the remaining 6 cases (Table 2). The merosin stain, which visualizes an integral component of the basal membrane, similarly showed unstained segments, or indicated splitting of the membrane, in 6 cases. Thirteen invaded fibres stained for fast and 17 for slow myosin heavy chain, while 10 stained with both. In addition to CD8⁺ cells, granzyme B⁺ cells were found in all invading infiltrates (Table 3). The infiltrates also contained an abundance of CD68⁺ cells, and at least a few CD163⁺ cells. The latter were however mainly located outside the fibre. One of the 20 partially invaded fibres was Bcl-2⁺, while 8 were FAS⁺ and 9 HSP70⁺ (Table 3). In order to illustrate some of the different staining patterns of partial invasion, 3 cases are presented in Fig 3.

Table 3. Characterization of partial invasions (pi).

Case	Pi	Bcl-2	FAS	Hsp70	Fast	Slow	Spectrin	Merosin	Granzyme B	CD68	CD163
1	1	−	+	−	+	+	+	−	+	+	+
	2	−	−	+	−	+	+	−	+	+	+
	3	+	+	−	+	+	+	−	+	+	+
	4	−	+	+	+	+	+	−	+	+	+
2	5	−	+	+	+	+	+	−	+	+	+
	6	−	−	+	−	+	+	−	+	+	+
3	7	−	−	−	−	+	+	+	+	+	+
	8	−	−	−	−	+	−	+	+	+	+
	9	−	+	+	+	−	−	+	+	+	+
4	10	−	−	−	+	−	−	+	+	+	+
5	11	−	−	+	+	+	+	+	+	+	+
	12	−	−	+	+	+	+	+	+	+	+
	13	−	+	+	+	+	+	+	+	+	+
6	14	−	−	+	+	−	−	+	+	+	+
7	15	−	−	−	−	+	−	+	+	+	+
8	16	−	−	−	−	+	+	+	+	+	+
	17	−	+	−	+	+	+	+	+	+	+
	18	−	−	−	−	+	+	+	+	+	+
9	19	−	−	−	+	+	+	+	+	+	+
10	20	−	−	−	+	+	−	+	+	+	+

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There were 20 partial invasions in the imaged muscle section areas (1.37 mm²) of the 10 cases.

The staining characteristics are shown as positive (+), or negative (-), representing stained or unstained invaded fibres or inflammatory cells. For spectrin and merosin, (+) denotes entire staining and (-) the presence of unstained segments of the membrane.

Fig 3 — Three cases (1–3) of partial invasions are shown (stained with haematoxylin-eosin (HE), MHC I, CD8, granzyme B, spectrin, merosin 80, CD68 and CD163. Case 1 and 2 have broad based partial invasions in the HE stained sections (case 1-2a), while case 3 has a branching appearance of invading cells (Case 3a). The invaded fibres show sarcoplastic staining of MHC I (case 1-3b). Several invading cells are CD8⁺ (case 1-3c) and the infiltrate contains granzyme B⁺ cells (case 1-3d). The cell membrane is pushed forward into the fibre by the invading cells, as seen by the spectrin stain (case 1-3e), but cases with discontinuity of cell membrane staining are also found (exemplified by case 3e, arrow). The inflammatory cells traverse the basal membrane (case 1f, arrow), which often seems to retain the original form of the fibre (case 2-3f), but is in some cases split (1-2f). There are CD68⁺ cells (case 1-3g) among the invading cells. CD163⁺ cells are seen in the infiltrate, but mainly outside the fibre (case 1-3h).

Expression of MHC I but never FAS in fibres with apoptotic nuclei

Within the selected areas of two TUNEL stained sections, separated only by the negative control section (6 μm), there were 8 muscle fibres with a TUNEL⁺ nucleus found (in 5 of the pi-patients) that could be confirmed to be inside the fibre membrane, after re-staining with spectrin (Table 4). This corresponds to a mean of 0.22 TUNEL⁺ myonuclei per 100 counted fibres (i.e. fibre sections). The purpose of the additional stains of the fibre and the surrounding inflammatory cells was to elucidate a possible apoptosis inducing pathway, as well as identifying protective or inducing factors. All 8 fibres had up-regulated MHC I in the sarcolemma and the sarcoplasm. None of these fibres stained for FAS, but one for Bcl-2 and 5 for HSP70. All 8 fibres had CD8⁺, CD68⁺ and CD163⁺ and 7 had granzyme B⁺ cells, adhering to the sarcolemma (Table 3). In the remaining case, the closest detected granzyme B⁺ cell was approximately 20μm from the fibre membrane. Notably, 2 fibres showed single CD8⁺ and granzyme B⁺ cells inside the cell membrane, close to a TUNEL⁺ myonucleus. Two examples of fibres with a TUNEL⁺ myonucleus together with stains of selected marker proteins are shown in Figs 4 and 5. No signs of necrosis, vacuolisation or classical partial invasion were found in these fibres. Moreover, spectrin staining showed no unstained section of the cell membranes of these fibres, which all stained negative for MAC. No TUNEL⁺ fibre nucleus was detected in the selected areas of DM patients or controls. However, TUNEL⁺ inflammatory cells (S2A and S2B Fig) were detected in both pi- and DM-patients, with a mean of 0.62 and 0.66 per 100 fibres, respectively, but not in the controls.

Table 4. Immunohistochemical characterization^* of fibres with apoptotic nuclei (TUNEL⁺) and adherent inflammatory cells.

No	MHC1	MAC	Bcl-2	Hsp70	FAS	Fast	Slow	CD8	Granzyme B	CD68	CD163
1	+	−	−	+	−	+	+	+	+	+	+
2	+	−	−	+	−	−	+	+	+	+	+
3	+	−	−	+	−	−	+	+^**	+^**	+	+
4	+	−	+	+	−	−	+	+	+	+	+
5	+	−	−	−	−	−	+	+	+	+	+
6	+	−	−	−	−	−	+	+	+	+	+
7	+	−	−	+	−	−	+	+	− #	+	+
8	+	−	−	−	−	+	−	+^**	+^**	+	+

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*i e expression (+) or no expression (–),

**CD8⁺ and granzyme B⁺ cells located inside the fibre membrane, # nearest detected granzyme B⁺ cell located 20 μm from the fibre.

Fig 4 — A TUNEL⁺ nucleus* is shown (a, arrow), which has a morphology of a myonucleus and is located inside the sarcolemma (b). The sarcoplasm of the fibre stains for MHC I (c), but not MAC (d). CD8⁺ cells are seen inside the fibre (e, arrows), as is one granzyme B⁺ cell (f, arrow), which is located close to the TUNEL⁺ myonucleus. The HE stained sections (g,h) adjacent to the above sections (a-f) do not show signs of classical partial invasion (or necrosis). *Fragmented DNA identifying an apoptotic nucleus stains brown, whereas not affected nuclei stain green (Methyl green).

Fig 5 — A fibre with a TUNEL⁺ nucleus (a, arrow), located inside the cell membrane (b), is shown. The fibre has CD68⁺ (c) and CD163⁺ cells (d) adhering to the sarcolemma (arrows). The fibre does not stain for Bcl-2 (e), FAS (f) or fast myosin heavy chain (g), but for slow myosin heavy chain (h).

Apoptotic nuclei are found almost exclusively in IIM with partial invasion

In order to provide a more reliable basis for the quantification of TUNEL⁺ fibre nuclei, the entire area of the two TUNEL-stained sections of all patients were studied by light microscopy. The number of fibres in the sections ranged from 954 to 3669 in the pi-patients, from 1019 to 3910 in the DM-patients and from 864 to 2968 in the controls. Forty-five TUNEL⁺ nuclei were unevenly distributed among all 10 pi-patients (range 1–13), compared to 4 in 3 of the DM-patients and none in the controls. These TUNEL⁺ nuclei were judged as being fibre nuclei based by their localisation and morphology, but were not confirmed by spectrin re-staining of the fibre membrane (S2 Fig), as were the TUNEL⁺ myonuclei in the selected areas. In the DM-cases, 2 of the 4 TUNEL⁺ fibres were found in a section which also contained a (sic) partial invasion. In 3 pi-cases a fibre with two apoptotic nuclei was found in the same section (exemplified in S2D Fig). No TUNEL positivity was found in the necrotic fibres of the selected areas. However, when studying the complete sections, rare necrotic fibres showed different grades of diffuse sarcoplasm staining (S3 Fig), whereas others only showed TUNEL⁺ inflammatory cells invading the fibre (S3 Fig).

Discussion

The presence of apoptosis in IIM has long been debated and its pathogenic role in these diseases considered unlikely [22]. Using the TUNEL assay we detected apoptotic muscle nuclei in inflammatory myopathies, which is in line with earlier studies [20, 21]. Other studies, using similar methods, did not detect apoptosis in IIM (or an increase, compared to controls) [19, 30, 48–50]. These studies, however, showed important differences, compared to our study, regarding presence of partial invasions, size of investigated area and main focus of the study, which may explain this discrepancy.

Interestingly, our results showed that apoptotic nuclei were present almost exclusively in biopsies containing partial invasion, and that the presence of apoptosis was strongly associated with MHC I expressing muscle fibres that did not show the classical signs of partial invasion. However, these fibres did have adherent inflammatory cells expressing CD8⁺, granzyme B⁺ as well as CD68 and CD163. The two major theories of how granzyme B gain entry into the cytosol of the target cell differ in respect of whether a passive transfer via pores (perforin) is possible or an active transport in necessary [51]. The identification of CD8⁺ and granzyme B⁺ cells in the sarcoplasm in 2 of 8 cases in this study suggests yet another in vivo mechanism of granzyme-target cell entry. Taken together, our findings lend support to an immune-mediated mechanism leading to apoptosis. This process seems to occur in muscle affected by partial invasion, but apparently constituting a parallel process. We would like to point out that double staining of the inflammatory cells was not possible, which means that it cannot be ruled out that NK-cells, although rare in endomysial infiltrate [6], may be represented as some of the granzyme B⁺ cells. Further, other pathological subgroups of IIM than those with partial invasion and pathology characteristic of DM were not studied. The presence or absence of apoptosis in the IIM subgroups classified as immune-mediated necrotizing myopathy, unspecific myositis and the recently emerged group characterized by perifascicular pathology, associated with an antisynthetase syndrome [52] should be addressed in further studies.

The absence of FAS in fibres with apoptotic nuclei, which is in agreement with earlier studies [16, 19, 30], together with the presence of granzyme B⁺ cells, strongly favours a cytotoxic mode of apoptosis induction rather than a FAS-mediated mechanism. Other proteins secreted by the CD8⁺ cytotoxic T cells, such as other granzymes [53] or other molecules [15, 54] may be alternative, albeit less likely, candidates of inducing apoptosis.

Apoptotic nuclei have earlier been demonstrated in muscle from IBM patients using sequential stains [20]. As pointed out by the authors of this report, the presence of single apoptotic nuclei in muscle fibres does not per se prove a general apoptotic process in the fibre. In addition, the low number of apoptotic fibre nuclei in IIM has been interpreted as lack of pathogenic relevance [20, 22]. However, the short time window of DNA fragmentation detected by TUNEL-staining, in the range of 1–3 hours, suggests that its expected appearance in normal muscle would be a rare finding [45]. Therefore, our finding of 3 fibres with 2 TUNEL⁺ myonuclei in the same section is an event that unlikely would occur by chance only, and indicates that the apoptotic process may be affecting a segment of the fibre.

Our finding (mean of 0.22 TUNEL⁺ myonuclei per 100 fibres) is within the range of earlier studies detecting apoptotic myonuclei in IIM, with means calculated from presented data from 0.079 [20] to 0.36 [21]. The differences in frequency may be accounted for by of different methods, and by the areas examined, in our case the area surrounding partial invasion. In line with other studies [8, 50], we did not detect apoptotic myonuclei in partially invaded myofibres. Notably, in the study of Hutchinson et al. [20], investigating PM and IBM, 2 of 100 fibres with TUNEL⁺ myonuclei were found in a partial invasion. Constituting such a small fraction, it was however concluded that apoptosis is an unlikely fate of such affected fibres. This conclusion agrees with our findings, indicating that apoptosis may be a parallel form of fibre degeneration, aside from the fibre disintegration seen in partial invasion. The rarity of observed apoptotic myonuclei in IIM, in spite of a repeatedly reported presence of granzyme B and perforin [14, 15], has been attributed to presence of anti-apoptotic proteins in myofibres, such as Bcl-2 [22]. An alternative explanation could be properties of the cell membrane of myofibres, impeding the entry of granzyme B, or granzyme B secreting lymphocytes, which were observed in two TUNEL⁺ fibres in this study.

The landmark investigations by Arahata and Engel [6, 55], using immunohistochemistry, showed that the majority of the invading cells of partial invasion are CD8⁺ cytotoxic cells, followed in frequency by CD68⁺ macrophages. Ultrastructural studies by the same authors [8] showed that the infiltrating cells push the morphologically intact cell membrane into the fibre, and although membrane reparative signs where noted, classical signs of fibre necrosis or apoptotic nuclei were not observed. The immunohistological findings in our study generally agree with this description. However, the detection of sections of cell membrane, lacking spectrin in 6 of 20 cases, is likely to signify a disturbed cell membrane function, facilitating the sarcoplasmic entry of molecules present in the invading infiltrate. Consecutive HE-stained sections of partial invasions showed that the invading inflammatory cell infiltrate, apparently surrounded by a matrix, often also invaded neighbouring fibres, indicating an acquisition of an invasive potential of the infiltrate. Such invasive potential is likely triggered by factors in the microenvironment of the infiltrate [56]. Granzyme B, which also may be produced by myeloid dendritic cells [57], has several substrates in the extracellular matrix [57] and is able to cleave and activate several pro-inflammatory cytokins [58]. Thus, granzyme B may, apart from its pro-apototic effect, also affect the properties, such the invasiveness, of the infiltrate.

The macrophages present in IBM and PM have been reported to express the BDCA-2 antigen and show a branching morphological appearance, typical of myeloid dendritic cells [24]. Here, this morphological appearance was noted in the CD68 and CD163 stains as well. It is unknown if these cells, with macrophage and myeloid dendritic properties, are able to activate T cells locally by presenting antigen [24], and if they secrete directly acting inflammatory molecules [25]. However, these cells are likely important in the muscle specific form of immune attack studied here. In this study, the CD163⁺ macrophages constituted a small fraction of all macrophages (CD68⁺) in control muscle, but their relative ratio to CD68⁺ macrophages was increased in inflammatory myopathies. Further studies, using multiple cell population and activation markers, may help to clarify the immunologic function of these cells, with seemingly overlapping macrophage and myeloid dendritic cell properties. Concomitant immune labelling of cells in muscle and blood from patients may further clarify if CD163⁺ macrophages are recruited to counteract the inflammatory reaction [26], or are a result of a type switch of present M1 macrophages in muscle during the disease process.

Bcl-2 expression has been shown to protect against different kinds of cell death [59–62], including pathways mediated by granzymes [34]. The present findings of a constitutive expression of Bcl-2 in healthy muscle, and a lower expression of Bcl-2 in IIM, confirm results from our previous study [18] and the study by Ibi et al. [63]. The Bcl-2 expression of the pi-cases in the present study was similar to the expression found in polymyositis patients in our previous study [18], with a median of 7.8 and 5.0%, positive fibres, respectively [18]. Notably, in the present study, the controls (median age 46 yrs.) showed clearly lower expression compared to the healthy volunteers (median age 28 yrs.), in the previous study, with Bcl-2 median values of 38% and 91%, respectively. This agrees with a reported lower expression of Bcl-2 in healthy individuals with increasing age [64], which may affect the vulnerability of muscle towards IIM and other myodegenerative processes. We observed one fibre with a TUNEL⁺ nucleus, which also expressed Bcl-2, indicating that its expression does not preclude an apoptotic process in muscle fibres. HSP70 expression was absent or faint in most control patients, but higher in the majority of patients with inflammatory myopathies, particularly in pi-patients. However, the great variation within the groups makes interpretation difficult, other than that, HSP70 is unlikely to have a specific role in the two studied types of fibre degeneration.

In conclusion: Apoptosis of muscle fibre nuclei is present in inflammatory myopathies with partial invasions, and these processes occur in the same area, but rarely in the same fibre. The affected fibres express MHC I and are surrounded by CD8⁺ T-cells, macrophages and granzyme B⁺ cells. The findings collectively support that apoptosis, induced by cytotoxic CD8⁺ T-cells in IIM, may be a mechanism activated in parallel with the fibre disintegration seen in partial invasion. In addition to its apoptosis inducing potential, granzyme B may have other important roles in IIM with partial invasion. Inflammatory cells with macrophage/myeloic dendritic properties are also present in infiltrates invading muscle fibres and adhere to fibres with apoptotic myonuclei.

Supporting information

S1 Dataset

(XLS)

Click here for additional data file.^{(51KB, xls)}

S1 Fig. The morphology of macrophages in IIM.

The stains of the CD68⁺ and the CD163⁺ cells in the cases with partial invasion (a) and dermatomyositis (b) indicate an expanded cytoplasm, compatible with a dendritic morphology, which is not seen in the non-inflammatory control cases (c and d [close-up]).

(TIF)

Click here for additional data file.^{(1MB, tif)}

S2 Fig. TUNEL-stain of inflammatory cells and myoneclei.

Two examples of TUNEL⁺ inflammatory cells (arrows) are shown, one close to a partial invasion with a branching appearance (a) and one in a case with dermatomyositis (b). When investigating the whole sections of the cases with partial invasion 45 TUNEL⁺ myonuclei were found, as judged by their morphological appearance and location, as exemplified here (c). One of the 3 cases in which 2 TUNEL⁺ myonuclei in the same fibre where found is shown (d).

(TIF)

Click here for additional data file.^{(1.4MB, tif)}

S3 Fig. TUNEL-stain of necrotic fibres.

The vast majority of the necrotic fibres showed no TUNEL-stain (a). But rare necrotic fibres did show varying degrees of diffuse sarcoplasmic stain (b,c). The nuclei of some invading inflammatory cells stained TUNEL positive (d).

(TIF)

Click here for additional data file.^{(1.2MB, tif)}

Acknowledgments

We thank Erik Edston for supplying us of with heart tissue as a positive control for the TUNEL-method.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

OD received a finacial grant from Region Östergötland. Grant no: LIO-535621. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

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Partial invasion and apoptosis in idiopathic inflammatory myopathies; parallel processes mediated by CD8+ cytotoxic T cells

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: Olof Danielsson et al, analysed muscle biopsies from 10 IIM patients, 10 DM patients and 10 donors without muscle disease. They performed TUNEL assays and immunohistochemistry on muscle biopsies. The authors have four main conclusions :

1. IIM is associated with alteration in the expression of Bcl-2, FAS, HSP70, CD163 and CD68.

2. Apoptotic nuclei express MHC I but not FAS

3. Apoptotic nuclei are found almost exclusively in IIM with partial invasion

4. Majority of invading cells in partial invasions are CD8+ cytotoxic cells

Authors suggest that partial invasion and apoptosis are likely mediated by cytotoxic CD8 cells

Major comments :

1. Title of the manuscript is « Partial invasion and apoptosis in idiopathic inflammatory myopathies; parallel processes mediated by CD8+ cytotoxic T cells », nevertheless the authors have only 10 cases of IIM, including 3 IBM def (1 poss), 1 overlap def, PM….. Following the 119th ENMC classification, IIM also include IMNM and DM. Why is DM placed separately in a different group, why is there no IMNM case. Sample size is a little small to conclude.

2. Line 299 : « In the DM-cases, 2 of the 4 TUNEL+ fibres 300 were found in a section which also contained a (sic) partial invasion. ». How many DM patients have partial invasions.

3. Line 190 : « IIM is associated with alteration in the expression of Bcl-2, FAS, HSP70 and of CD163/CD68 ratio ». Authors could consider changing this sentence, as no significant differences in the expressions of FAS and HSP70 are observed between DM and controls (DM are IIM).

4. Table 1 : Authors show necrotic fibres in 4 out of 10 patients. Surprisingly necrotic fibres are not TUNEL+ fibres and vice versa. It has been described that TUNNEL assay does not discrimate apoptosis from necrosis (BETTINA GRASL-KRAU et al, HEPATOLOGY, May 1995). How can the authors explain these findings.

5. The lack of expression of FAS in apoptotic nuclei is indeed surprising. Nevertheless, considering the relatively low mean frequency of TUNEL+ myonuclei ( 0.22 per 100 fibres) and the presence of TUNEL+ fibres in only 5 out of 10 patients, it is possible that these TUNEL+ myonuclei do not have any pathological significance. A greater sample size could help make the results more robust.

6. Authors show increase frequency of FAS+ fibers in pi-patients compared to controls, nevetheless apoptotic fibers do not express FAS. How can the autors explain this, do non-apoptotic fibers express FAS.

7. Line 99 : control samples are young compared to pi-patients, mark difference in age distribution.

Minor comments :

1. Table 1 : Patient 7, change « IMB » to « IBM »

2. How can the authors confirm that partial invasion and apoptosis are mediated by CD8 cytotoxic cells. Can the authors show co-immunostaining of CD8 and granzyme B.

3. Line 231 : « In addition to CD8+ cells, granzyme B+ cells were found in all invading infiltrates (Table 2). » Table 2 shows Antibodies and immunohistochemical procedure, maybe the authors wanted to state Table 3 ?

Reviewer #2: Authors performed immunohistochemical examinations of muscular tissues which were treated with some biological markers including phenotypical, cytotoxic, or enzymatic protein, and apoptosis assay; in addition, they were statistically analyzed between idiopathic inflammatory myopathies (IIM) including polymyositis (PM) or inclusion body myositis (IBM) and controls including dermatomyositis and others. This manuscript has some issues to describe as follows.

１. Turkey’s multiple comparison test is usually performed in the parametric population. In this study, is it appropriate to use Turkey’s test? Otherwise, extracted data should be re-analyzed by the non-parametric statistics.

２. In the figure 1, CD163/CD68 ratio was shown. Can you explain what the frequencies of the double positive cells with CD68 and CD163 were expressed?

３. Authors should indicate the clinical findings related to the musculoskeletal impairment in all participants.

４. It is important to clarify the implication of the pathological characteristics in IIM through showing the correlation between clinical findings and analyzed data based on the basic research. Please demonstrate how the immunopathological findings shown in this study are implicated in the development of clinical impairment. Namely, authors should indicate the relationship between laboratory data/muscular scores and the immunopathological data which were detected in this study. Laboratory data may include serum creatine kinase and/or aldolase, and muscular scores may be manual muscle test (MMT). MMT8 is useful for scoring muscle weakness as the prevalent evaluation of IIM.

５. What kinds of autoantibodies were related to the patients with IIM in this study? Recently, IIM has been recognized as the overall category; moreover, each patient should be categorized into each classification of inflammatory myositis according to the disease-specific antibody. If possible, it is ideal to show the relationship between specific antibody and analyzed data.

６. Immuno-mediated necrotizing myopathy (IMNM) should be differentiated from other types of PM or IBM. Author should demonstrate the differences of characteristics between IMNM, other types of PM, and IBM, if applicable.

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2020 Sep 16;15(9):e0239176. doi: 10.1371/journal.pone.0239176.r002

Author response to Decision Letter 0

23 Jun 2020

The responses are uploaded as a separate file.

Alfred S Lewin, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements:

We have done our best to comply with the style requirements, but will certainly make any necessary additional corrections.

We rewrote the first two paragraphs in the methods section, and added a third, with more detailed information about the recruitment process and the representativeness of the included cases (line 97-141, below), and we added demographic details of all participants in the S1 Table (please see uploaded file).

Methods

Patients and biopsies

“All biopsies were taken from the anterior tibial muscle for diagnostic purposes. We included stored tissue samples, obtained 1997-2002, with confirmed partial invasion from 10 patients (median age 62 years, range 32-75) with inflammatory myopathies (Table 1), which in a previous study [4] had been classified according to the Amato/European neuromuscular centre (ENMC) classification [7]. These samples were compared with two control groups, comprising 10 patients in each group. As an IIM control group, biopsies from patients with DM were selected; the reason for this choice was that this disease has another type of, more or less pathognomonic, pathological findings [7]. The DM cases (median age 52 years, range17-78) were consecutive biopsies investigated in our lab, where the available clinical and laboratory data allowed the classification of at least probable DM, according to the Amato/ENMC. Eight cases had a definite and two a probable DM according to this classification, and all cases had a definite DM according to the Bohan and Peter. The other control group comprised consecutive cases (median age 46 years, range 39-59), where neuromuscular disease had been excluded, based on biopsy and clinical examination. The cases in these latter two groups were included during April 2015. For demographic details of all cases, see S1 Table.

Sectioning method and selection of cases with partial invasion

In the earlier study [4], muscle biopsies from 36 patients were shown to contain at least one partial invasion. In order to detect and verify strict biopsy criteria stated by Amato/ENMC [7], 35 consecutive cryosections had been made from each biopsy, and every 7th section (6 µm) stained with haematoxylin eosin (HE). Partial invasions were first identified by the morphologic appearance in the HE stained sections and then confirmed by the following three sections, stained with antibodies against MHC I, CD8 and membrane attack complex (MAC). MAC was included to add sensitivity to detect myofibre necrosis [43]. For the present study, the frozen consecutive sections (-70º C) of the biopsies were thawed, and the HE stained sections were again studied in a Zeiss AXIO Imager light microscope. From the 36 cases, we selected those where the partial invasion were present in at least 3 HE sections (n = 10), and used the intermediate sections for immunohistochemistry and the TUNEL-assay.

Reflections on the representativeness of the included cases

The requirement that the partial invasion had to be detectable in 3 HE stained sections, had the effect that partial invasions with a smaller size than approximately 150 µm were not included. We have not found any report voicing the possibility that partial invasions of different sizes may differ in a qualitative manner, but it cannot be excluded. We earlier showed that partial invasions is present in many types of IIM [4]. However, it is more common and more frequent in IBM, followed by PM [44]. The distribution of diagnoses of investigated pi-cases is comparable with that found in the 36 cases in our 6 year IIM cohort [4] (Table 1). With the above reservation of pi-size, the results are thus considered representative for IIM with pi, not of any defined disease subgroup.”

Reviewer 1

1. IIM is associated with alteration in the expression of Bcl-2, FAS, HSP70, CD163 and CD68.

2. Apoptotic nuclei express MHC I but not FAS.

3. Apoptotic nuclei are found almost exclusively in IIM with partial invasion

4. Majority of invading cells in partial invasions are CD8+ cytotoxic cells

Authors suggest that partial invasion and apoptosis are likely mediated by cytotoxic CD8 cells

Major comments :

The presence of partial invasion (pi), according to strict criteria, was our method to identify IIM-cases, with an immune response characterized by CD8+ cytotoxic T cells. The reason for choosing dermatomyositis, for comparison, was that in this disease (or group of diseases) the immunological response is different, and only rarely includes partial invasion. Immune-mediated necrotizing myopathy is pathologically dominated by necrosis, and, in the majority of cases, the complete absence of inflammatory infiltrates. We were mainly interested in apoptosis-inducing mechanisms induced by inflammatory cells, and although plausible hypotheses have been presented, the immunopathology of IMNM is at this point less clear (1, 2), thus we decided to not include IMNM. We have now added a comment of IMNM in the introduction section (line 55-61):

“Another widely accepted pathology based subgroup of IIM is immune-mediated necrotizing myopathy (IMNM), also called necrotizing autoimmune myopathy (NAM) (3) [in text ref. 7]. There is an ongoing discussion about further myopathological subdivisions of these diseases (4, 5) [in text 5, 9]. However, partial invasion is a feature that is common to both IBM and PM (strictly defined), and extensive sectioning of muscle tissue shows that partial invasion is sometimes encountered also in overlap syndromes, and, rarely, in classical DM (6, 7) [in text 4, 6]. This raises the question whether there are further shared pathogenic processes associated with the feature of partial invasion.”

As a response of the editor’s requirement of additional information about the participant recruitment method and the demographic details of your participants, we rewrote the first two paragraphs in the methods section, and added a third. We included a comment explaining our choice of IIM control group in the method section (line 101-103).

“As an IIM control group, biopsies from patients with DM were selected; the reason for this choice was that this disease has another type of, more or less pathognomonic, pathological findings (3) [in text 7].

We think that there is much evidence supporting the view that there are more than one pathologic process present, not just in IBM, but also in the other IIM. Also, several non-immune mechanisms have been suggested (8, 9). The studies of Arahata and Engel (7, 10) showed typical immunopathology of the different IIM, but no absolute disease correlation. Our earlier study highlighted this pathological overlap (6), which may be of importance when using pathology in classifications of IIM. In this study our focus was the pathological process with a known potential to induce apoptosis, rather than a classified disease entity.

In response to the editor’s urge, for a statement about the representativeness of our sample) in the method section, we added a paragraph, where we explained this more clearly (line 135-141):

“The requirement that the partial invasion had to be detectable in 3 HE stained sections, had the effect that partial invasions with a smaller size than approximately 150 µm were not included. We have not found any report voicing the possibility that partial invasions of different sizes may differ in a qualitative manner, but it cannot be excluded. We earlier showed that partial invasions is present in many types of IIM (6) [in text 4]. However, it is more common and more frequent in IBM, followed by PM (11) [in text 44]. The distribution of diagnoses of investigated pi-cases is comparable with that found in the 36 cases in our 6 year IIM cohort (6) [in text 4] (Table 1). With the above reservation of pi-size, the results are considered representative for IIM with pi, while not of any defined disease subgroup.”

Regarding the number of cases: The discussion about the significance of partial invasion in diagnosing IIM, and the controversy of PM as a diagnostic entity (12-14), emphasized the need of using strict criteria for partial invasion. This resulted in a modest number of cases, however similar to earlier studies. Although the detected TUNEL+ myonuclei in the circled areas of pi-patients clearly contrasted to the absence in DM-cases, we felt, as pointed out by the reviewer, that more data would be desirable. That was the reason why the whole sections were studied for TUNEL+ myonuclei, (line 303-306):

“In order to provide a more reliable basis for the quantification of TUNEL+ fibre nuclei, the entire area of the two TUNEL-stained sections of all patients were studied by light microscopy. The number of fibres in the sections ranged from 954 to 3669 in the pi-patients, from 1019 to 3910 in the DM-patients and from 864 to 2968 in the controls.”

As the method of detecting positive myonuclei differed, in the way that only those myonuclei with typical morphology and a location clearly inside the sarkolemma were accepted (S2 Fig. c,d) in this extended search, we chose to present these results in a separate paragraph.

We think that the similar frequency of detected apoptosis as in earlier studies, the almost complete absence of TUNEL+ myonuclei in DM and the colocalization with granzyme+ cells, lend support to our conclusion.

2. Line 299 : « In the DM-cases, 2 of the 4 TUNEL+ fibres 300 were found in a section which also contained a (sic) partial invasion. ». How many DM patients have partial invasions.

In these 10 DM cases, this was the only one. Athough considered rare, it was already described by Arahata and Engel (7), and earlier noted by us (6).

We agree. The sentence has been changed (line 203-204): “IIM is associated with alteration in the expression of Bcl-2 and of CD163/CD68 ratio”.

We are grateful for this remark. We missed to attach the third supplementary figure file (S3 Fig), now added, which is relevant for this question (see below).

As a consequence of the findings of Grassel-Kraupp et al., Edston et al. (15), who generously supplied us with tissue for positive control, investigated decomposing heart tissue with two different TUNEL-kits. They found that false TUNEL positivity can occur in autolytic fibres with the Cardiotacs kit (due to inadequate fixation), but not with Apotag kit (which we used).

The referred studies, investigating apoptosis in muscle, do not report TUNEL-positivity of necrotic fbres. Olivé et al. specifically state that no necrotic myofibres stained by DNA fragmentation in situ labeling of muscle tissue (16). However, in this study we show that rare necrotic fibres do show TUNEL-stain diffusely in the sarcoplasm (S3 Fig).

Typical morphologic findings of fibre necrosis are detectable also in the TUNEL-stain of TUNEL- negative fibres, and the stains of the adjacent sections are well suited to detect necrotic fibres (especially the HE and spectrin stains). The reason for including MAC (C5-C9), was to detect supple signs of necrosis. Engel and Biesecker concluded that complement activation is an “invariant concomitant of muscle fibre necrosis” (17). To draw attention to this we have added in the methods section (line 128): “MAC was included to add sensitivity to detect myofibre necrosis (17).”

5. The lack of expression of FAS in apoptotic nuclei is indeed surprising. Nevertheless, considering the relatively low mean frequency of TUNEL+ myonuclei (0.22 per 100 fibres) and the presence of TUNEL+ fibres in only 5 out of 10 patients, it is possible that these TUNEL+ myonuclei do not have any pathological significance. A greater sample size could help make the results more robust.

For the comment on FAS, please see comment 6.

For the comment on robustness, please see comment 1.

The circled and photographed areas had been chosen because they contained the pathogenic process of interest and allowed these fibres to be studied in all, differently stained, sections. This allowed the frequency of the different pathological findings to be compared. In this area, there were 20 partial invasions, 5 necrotic fibres and 8 TUNEL+ myofibres and 21 TUNEL+ inflammatory cells. When comparing the relative frequency of these finding, one has to consider:

1. The apoptotic myonuclei were studied in 2 sections.

2. The TUNEL+ myonuclei are somewhat larger and inflammatory cells are somewhat smaller than the size of one section, and are as a rule, not seen in neighbouring sections.

3. Necrotic fibres and partial invasions are of far greater size, and seen in many sections. A requirement for partial invasionen in this study was that it could be detected in 21 consecutive sections.

4. The time interval, during which a fibre necrosis can be detected is longer than that of a TUNEL+ nucleus (Karpati and Molnar estimated 6-8 hours, until the invasion of phagocytic macrophages (18)), and we estimate that the time process of partial invasion is as least as long. If these assumptions are sound, apoptotic myonuclei are more prevalent than necrosis and partial invasion. This would be reasonable, as myonuclei are plentiful and only serve a limited sarcoplasmic domain.

The question of pathological significance of these signs of muscle fibre demise is relevant, but also applies to other observed pathological changes found in IIM. There is, overall, a dissociation between inflammation and the clinical finding of muscle weakness in IIM (6, 19) and parallel non-inflammatory mechanisms have been identified (20). We think that there are several missing pieces in our understanding of these diseases, and that our findings of myonuclear apoptosis, being present in a pathologically defined subgroup of IIM, but not in the control groups, deserves attention and further study.

6. Authors show increase frequency of FAS+ fibers in pi-patients compared to controls, nevertheless apoptotic fibers do not express FAS. How can the autors explain this, do non-apoptotic fibers express FAS.

The expression of FAS in non-apoptotic fibres has been shown in earlier studies (ref. line 335-336), and different explanations have been put forward. Behrens et al. noted a co-expression of FAS and Bcl-2 and suggested a protective role of Bcl-2 (21). In an earlier study (22), we noted that, although both proteins were more commonly expressed on Type 2-fibres, the number of fibres expressing Bcl-2 was inversely related to the number of FAS expressing fibres. This is not evidence against a protective rule on a single fibre level, but it would require other means of apoptosis protection. De Bleecker et al. showed that most FAS+ fibres were NCAM-positive regenerating muscle fibres (23), and mention the function of FAS as a costimulatory factor in the generation of an immune response (24). They considered that a protective effect of Bcl-2 cannot be excluded, but suggested that muscle fibre FAS and Bcl-2 is part of an activated or reactivated regeneration program, that had “little to do with prevention or induction of apoptosis”. We have added this reference in the discussion section (line 335). In the review of Muscle fibre apoptosis in neuromuscular diseases, Dominique Tews also draws attention to the immune-activating role of FAS, and in addition to Bcl-2, mentions the upregulation other anti-apoptotic proteins as a possible explanation for the absence of apoptosis in FAS+ fibres (25).

We think that our findings with no TUNEL+ myonuclei in FAS+ fibres strengthen the conclusion that FASL-FAS does not induce apoptosis in IIM. One may consider the (perhaps remote) possibility of a time delay of apoptosis in FAS+ fibres, rarely observed because of its short time frame, but we think that our simultaneous observation of a granzyme B+ cell, in the sarcoplasm, next to an apoptotic myonucleus, in the absence of FAS, makes this very unlikely. We think that De Bleecker at al. (23) make a good case for their theory, and agree with the possible explanations put forward by Tews (25).

7. Line 99 : control samples are young compared to pi-patients, mark difference in age distribution.

Age distribution (years):

pi DM controls

median 63.5 57.3 45.3

SD 12.2 18.9 5.6

mean 61,7 52.3 46.0

As the reviewer points out, the age of the controls is lower than that of the pi-patient. To detect any age influence of the results we performed correlation analysis (Spearman) of the quantitative results within each group. In the control group without muscle disease, most correlation coefficients were close to zero. Only the HPS70 showed a positive correlation of 0.64 (p= 0.047). Here, an outlier with 30% HSP70+ fibres, was the main contributor (one case had 5%, two cases 1% the other six 0%). Had it been excluded, it would have been resulted in r 0.5578 and p= 0.1270. In the DM-group r was -0.12 (p= 0.72) and in the pi-group r was 0.22 (p=0.54).

No significant age correlation was found in the DM-group, where most of the correlations were close to zero.

In the pi-group there were statistically significant positive age correlations for three results: The percentage of FAS+ fibres (r 0.76, p= 0.015), percentage CD68+ cells (r 0.68, p= 0.03) and the percentage of fibres expressing fast myosin heavy chain (r 0.64, p=0.05). Our interpretation is that these correlations do not have any impact on our results. As the expression of these proteins showed very low r values in the other two groups, these correlations may represent type 1-errors.

We have also investigated the presence of TUNEL+ myonuclei in controls in the age groups 20-39 and 60-80 yrs in an ongoing study (not yet published), using the same method and setting (a circled area of the same size). No TUNEL+ myonucleus was found in these control groups.

Minor comments :

1. Table 1 : Patient 7, change « IMB » to « IBM »

Thank you, now changed.

2. How can the authors confirm that partial invasion and apoptosis are mediated by CD8 cytotoxic cells. Can the authors show co-immunostaining of CD8 and granzyme B.

The finding of granzyme B+ and CD8+ cells in the sarcoplasm, next to a TUNEL+ myonucleus in two cases was an unexpected finding, not described earlier. The cells stained by granzyme B and CD8 are not the same cells. The sections are on 18 and 12 µm, respectively, of either side of the TUNEL/spectrin stained section. It would indeed have been elegant to show a co-stain.

We applied a CD8 antibody, directly conjugated to fluorescent Alexa 488, to earlier stained granzyme B-stained sections, but the previous staining complex prevented binding of the earlier stained cells. Cytotoxic CD8+ and NK-cells are the only known inflammatory cells, known potential to induce apoptosis, mediated by granzyme B. NK-cells are rare in inflammatory infiltrates in IIM (7), but we cannot rule out that an NK-cell also may entered the sarcoplasm, and is the source of granzyme B. We have added a sentence in the discussion section to clarify this (line 331-334):

“We would like to point out that double staining of the inflammatory cells was not possible, which means that it cannot be ruled out that NK-cells, although rare in endomysial infiltrate (7) [in text 6], may be represented as some of the granzyme B+ cells.”

References

1. Preusse C, Goebel HH, Held J, Wengert O, Scheibe F, Irlbacher K, et al. Immune-mediated necrotizing myopathy is characterized by a specific Th1-M1 polarized immune profile. Am J Pathol. 2012;181(6):2161-71.

2. Allenbach Y, Arouche-Delaperche L, Preusse C, Radbruch H, Butler-Browne G, Champtiaux N, et al. Necrosis in anti-SRP(+) and anti-HMGCR(+)myopathies: Role of autoantibodies and complement. Neurology. 2018;90(6):e507-e17.

3. Hoogendijk JE, Amato AA, Lecky BR, Choy EH, Lundberg IE, Rose MR, et al. 119th ENMC international workshop: trial design in adult idiopathic inflammatory myopathies, with the exception of inclusion body myositis, 10-12 October 2003, Naarden, The Netherlands. Neuromuscular disorders : NMD. 2004;14(5):337-45.

4. De Bleecker JL, De Paepe B, Aronica E, de Visser M, Group EMMBS, Amato A, et al. 205th ENMC International Workshop: Pathology diagnosis of idiopathic inflammatory myopathies part II 28-30 March 2014, Naarden, The Netherlands. Neuromuscular disorders : NMD. 2015;25(3):268-72.

5. De Bleecker JL, Lundberg IE, de Visser M, Group EMMBS. 193rd ENMC International workshop Pathology diagnosis of idiopathic inflammatory myopathies 30 November - 2 December 2012, Naarden, The Netherlands. Neuromuscular disorders : NMD. 2013;23(11):945-51.

6. Danielsson O, Lindvall B, Gati I, Ernerudh J. Classification and diagnostic investigation in inflammatory myopathies: a study of 99 patients. The Journal of rheumatology. 2013;40(7):1173-82.

7. Arahata K, Engel AG. Monoclonal antibody analysis of mononuclear cells in myopathies. I: Quantitation of subsets according to diagnosis and sites of accumulation and demonstration and counts of muscle fibers invaded by T cells. Annals of neurology. 1984;16(2):193-208.

8. Henriques-Pons A, Nagaraju K. Nonimmune mechanisms of muscle damage in myositis: role of the endoplasmic reticulum stress response and autophagy in the disease pathogenesis. Curr Opin Rheumatol. 2009;21(6):581-7.

9. Manole E, Bastian AE, Butoianu N, Goebel HH. Myositis non-inflammatory mechanisms: An up-dated review. J Immunoassay Immunochem. 2017;38(2):115-26.

10. Engel AG, Arahata K. Monoclonal antibody analysis of mononuclear cells in myopathies. II: Phenotypes of autoinvasive cells in polymyositis and inclusion body myositis. Annals of neurology. 1984;16(2):209-15.

11. Arahata K, Engel AG. Monoclonal antibody analysis of mononuclear cells in myopathies. IV: Cell-mediated cytotoxicity and muscle fiber necrosis. Annals of neurology. 1988;23(2):168-73.

12. Amato AA, Griggs RC. Unicorns, dragons, polymyositis, and other mythological beasts. Neurology. 2003;61(3):288-9.

13. Chahin N, Engel AG. Correlation of muscle biopsy, clinical course, and outcome in PM and sporadic IBM. Neurology. 2008;70(6):418-24.

14. van der Meulen MF, Bronner IM, Hoogendijk JE, Burger H, van Venrooij WJ, Voskuyl AE, et al. Polymyositis: an overdiagnosed entity. Neurology. 2003;61(3):316-21.

15. Edston E, Grontoft L, Johnsson J. TUNEL: a useful screening method in sudden cardiac death. International journal of legal medicine. 2002;116(1):22-6.

16. Olive M, Martinez-Matos JA, Montero J, Ferrer I. Apoptosis is not the mechanism of cell death of muscle fibers in human muscular dystrophies and inflammatory myopathies. Muscle & nerve. 1997;20(10):1328-30.

17. Engel AG, Biesecker G. Complement activation in muscle fiber necrosis: demonstration of the membrane attack complex of complement in necrotic fibers. Annals of neurology. 1982;12(3):289-96.

18. Karpati G, Molnar, M. J. Muscle fibre regeneration in human skeletal muscle diseases. In: Schaffiano S, Partridge, T., editor. Skeletal Muscle Repair and Regeneration A A Dordrecht, The Netherlands: Sprnger; 2008. p. 200.

19. Dorph C, Englund P, Nennesmo I, Lundberg IE. Signs of inflammation in both symptomatic and asymptomatic muscles from patients with polymyositis and dermatomyositis. Annals of the rheumatic diseases. 2006;65(12):1565-71.

20. Coley W, Rayavarapu S, Pandey GS, Sabina RL, Van der Meulen JH, Ampong B, et al. The molecular basis of skeletal muscle weakness in a mouse model of inflammatory myopathy. Arthritis and rheumatism. 2012;64(11):3750-9.

21. Behrens L, Bender A, Johnson MA, Hohlfeld R. Cytotoxic mechanisms in inflammatory myopathies. Co-expression of Fas and protective Bcl-2 in muscle fibres and inflammatory cells. Brain : a journal of neurology. 1997;120 ( Pt 6):929-38.

22. Danielsson O, Nilsson C, Lindvall B, Ernerudh J. Expression of apoptosis related proteins in normal and diseased muscle: a possible role for Bcl-2 in protection of striated muscle. Neuromuscular disorders : NMD. 2009;19(6):412-7.

23. De Bleecker JL, Meire VI, Van Walleghem IE, Groessens IM, Schroder JM. Immunolocalization of FAS and FAS ligand in inflammatory myopathies. Acta neuropathologica. 2001;101(6):572-8.

24. Lynch DH, Ramsdell F, Alderson MR. Fas and FasL in the homeostatic regulation of immune responses. Immunology today. 1995;16(12):569-74.

25. Tews DS. Muscle-fiber apoptosis in neuromuscular diseases. Muscle & nerve. 2005;32(4):443-58.

Reviewer 2

We agree that non-parametric statistics are often a good choice for this type of data. Accordingly, for the results shown in the Figures 1a-e the non-parametric Kruskal-Wallis test was used, followed by Dunn´s multiple comparison test. However, the parametric ANOVA, followed by the Tukey´s test was chosen for the ratio CD163+/CD68+, as the deviation of the data from the mean (see below), conformed to a normal distribution, which was confirmed by the Pearson normality test. Ratios, as well as differences (delta values), may often be normally distributed even if the individual values are not. The statistical approach followed the advice from a statistician.

２. In the figure 1, CD163/CD68 ratio was shown. Can you explain what the frequencies of the double positive cells with CD68 and CD163 were expressed?

Since no double staining was done, we cannot say for sure that it is the same cell that expresses CD68 and CD163. However, CD68 is an established macrophage pan-macrophage marker, while CD163 is marker of a macrophage subpopulation. We therefore reported the ratio between the subpopulation and the “mother” population. The two populations were stained in adjacent sections (6 µm thick), thus we believe that our data are representative of the relative occurrence of type 2 macrophages.

３. Authors should indicate the clinical findings related to the musculoskeletal impairment in all participants.

We agree that it is relevant to clarify to the reader why two of the patients were classified as not IIM according to Amato/ENMC, but according to Bohan and Peter. In the legends to Table 1 (line 115-116) we added:

“# These two patients did not have a detectable muscle weakness and could thus not be classified according to Amato/ENMC.”

We think that the sample size in this study is too small, and the selection of samples not designed to draw any conclusion related to musculoskeletal impairment. In our earlier study (1), we showed that partial invasion (strictly defined) was not exclusively found in IBM and PM, but also in other IIM disease groups, and also found in patients with muscle pathology consistent with IIM, but without muscle weakness (e. g. overlap syndromes). Contrary to the traditional Bohan and Peter classification, the Amato/ENMC requires muscle weakness for IIM classification (except for amyopathic dermatomyositis) (2-4).

CK was above normal in all IIM-patients of this study, and was one of the criteria for classification. In the legends to Table 1 (line 115) we added: “* The creatine kinase was elevated in all patients.” We agree that MMT8 is a valuable and recommended outcome measure, as concluded by the 213th ENMC International Workshop (5), but the applied classifications, which were decisive for the selection of our cases, only define the pattern, not the degree of muscle weakness.

For this study, there were 19 muscle biopsies from the 10 pi-patients (1-3 per patient). From each patient the biopsy was chosen, which was best suited for classification. That means that the time from disease onset and the time of the analyzed biopsy varied, and in some cases treatment had been given. So we don´t think that any fair correlations can be done between pathology and clinical findings. The study was not designed for this purpose.

We agree that the advent of MSA and MAA has added a new and important dimension of IIM-classification. However, the focus of this study was the pathological findings considered more or less specific for IBM and PM. The Amato/ENMC classification, which served as a basis for this and our earlier study, does not require autoantibodies for diagnosis, and we did not obtain these data for inclusion. We also think that a different design of the study would have been necessary, to be able to draw any conclusion from the autoantibody status of the patients.

There were no patients with IMNM in this study. In IMNM inflammatory infiltrates are scarce and, when found, mostly perivascular. We have not found studies reporting partial invasion in IMNM, and we have so far not found partial invasion, fulfilling strict criteria, in this group (only partial necrosis). We therefore did not include IMNM patients in this study.

We realize that we had been too vague in our description. Therefore we rewrote the aim of the study (line 90-93), to clarify that our main focus was to investigate muscle from IIM patients with partial invasion, and use a pathologically likewise well defined group (DM) for comparison:

“The aim of the study was to investigate if apoptotic myonuclei are present in muscle in the specific subgroup of IIM with partial invasion, and to detect the presence of key molecules of the apoptotic-mediating or -protective pathways in the affected and in adjacent fibres. The CD8+ T-cell-mediated immune effector mechanism and frequency of type 2 macrophages were evaluated.”

We also clarified the rationale for selection of cases in the introduction section (line 55-61): “Another widely accepted pathology based subgroup of IIM is immune-mediated necrotizing myopathy (IMNM), also called necrotizing autoimmune myopathy (NAM) (6), characterized by muscle fibre necrosis and the relative absence of inflammatory cells. In addition, there is an ongoing discussion of further myopathological subdivisions of these diseases (7, 8). However, partial invasion is a feature that is common to both IBM and PM (strictly defined), and extensive sectioning of muscle tissue shows that partial invasion is sometimes encountered also in classical DM, as well as in overlap syndromes (1, 9). This raises the question whether there are other shared pathogenic processes, associated with partial invasion.”

1. Danielsson O, Lindvall B, Gati I, Ernerudh J. Classification and diagnostic investigation in inflammatory myopathies: a study of 99 patients. The Journal of rheumatology. 2013;40(7):1173-82.

2. Bohan A, Peter JB. Polymyositis and dermatomyositis (second of two parts). The New England journal of medicine. 1975;292(8):403-7.

3. Bohan A, Peter JB. Polymyositis and dermatomyositis (first of two parts). The New England journal of medicine. 1975;292(7):344-7.

4. van der Meulen MF, Bronner IM, Hoogendijk JE, Burger H, van Venrooij WJ, Voskuyl AE, et al. Polymyositis: an overdiagnosed entity. Neurology. 2003;61(3):316-21.

5. Benveniste O, Rider LG, Group EMOS. 213th ENMC International Workshop: Outcome measures and clinical trial readiness in idiopathic inflammatory myopathies, Heemskerk, The Netherlands, 18-20 September 2015. Neuromuscular disorders : NMD. 2016;26(8):523-34.

6. Hoogendijk JE, Amato AA, Lecky BR, Choy EH, Lundberg IE, Rose MR, et al. 119th ENMC international workshop: trial design in adult idiopathic inflammatory myopathies, with the exception of inclusion body myositis, 10-12 October 2003, Naarden, The Netherlands. Neuromuscular disorders : NMD. 2004;14(5):337-45.

7. De Bleecker JL, De Paepe B, Aronica E, de Visser M, Group EMMBS, Amato A, et al. 205th ENMC International Workshop: Pathology diagnosis of idiopathic inflammatory myopathies part II 28-30 March 2014, Naarden, The Netherlands. Neuromuscular disorders : NMD. 2015;25(3):268-72.

8. De Bleecker JL, Lundberg IE, de Visser M, Group EMMBS. 193rd ENMC International workshop Pathology diagnosis of idiopathic inflammatory myopathies 30 November - 2 December 2012, Naarden, The Netherlands. Neuromuscular disorders : NMD. 2013;23(11):945-51.

9. Arahata K, Engel AG. Monoclonal antibody analysis of mononuclear cells in myopathies. I: Quantitation of subsets according to diagnosis and sites of accumulation and demonstration and counts of muscle fibers invaded by T cells. Annals of neurology. 1984;16(2):193-208.

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PLoS One. doi: 10.1371/journal.pone.0239176.r003

Decision Letter 1

Alfred S Lewin

16 Jul 2020

PONE-D-19-35903R1

Partial invasion and apoptosis in idiopathic inflammatory myopathies; parallel processes mediated by CD8+ cytotoxic T cells

PLOS ONE

Dear Dr. Danielsson,

Reviewer 1 maintains that you have too few cases to draw your conclusions, and I agree with this assessment. This is the comment from the first review, " Title of the manuscript is « Partial invasion and apoptosis in idiopathic inflammatory myopathies; parallel processes mediated by CD8+ cytotoxic T cells », nevertheless the authors have only 10 cases of IIM, including 3 IBM def (1 poss), 1 overlap def, PM….. Following the 119th ENMC classification, IIM also include IMNM and DM. Why is DM placed separately in a different group, why is there no IMNM case. Sample size is a little small to conclude." Please either evaluate more patients or explain why you think we are incorrect.

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Reviewers' comments:

Reviewer's Responses to Questions

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Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

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Reviewer #2: Partly

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Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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6. Review Comments to the Author

Reviewer #1: Thank you for your clear and detailed responses. Nevertheless, i still believe that sample size is small to conclude, especially that myositis are a very heterogenous group of diseases, in addition to human inter-sample heterogeneity.

Reviewer #2: (No Response)

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PLoS One. 2020 Sep 16;15(9):e0239176. doi: 10.1371/journal.pone.0239176.r004

Author response to Decision Letter 1

23 Aug 2020

Response to reviewers (also uploaded as a separate file)

We agree that the number of cases is rather low, and that IIM is a heterogeneous entity. We therefore propose to change the title, so that it clearly shows that we have investigated a subset of IIM, namely IIM with partial invasion, thereby reducing the heterogeneity and highlighting the focus on a particular process. In addition, we have modified the text and conclusion, to clarify that we only study the phenomenon of partial invasion (which still is highly relevant) in a subset of IIM, and that verifying studies are needed to confirm the generalizability of our findings. Unfortunately, it would be difficult and take far too long time to include mores samples to the present paper.

The following changes of the manuscript have been made:

Former title:

Partial invasion and apoptosis in idiopathic inflammatory myopathies; parallel processes mediated by CD8+ cytotoxic T cells

New title:

Apoptosis in idiopathic inflammatory myopathies with partial invasions; a role for CD8+ cytotoxic T cells?

Added in Discussion, line 333:

Further, other pathological subgroups of IIM than those with partial invasion and pathology characteristic of DM were not studied. The presence or absence of apoptosis in the IIM subgroups classified as immune-mediated necrotizing myopathy, unspecific myositis and the recently emerged group characterized by perifascicular pathology, associated with an antisynthetase syndrome [52] should be addressed in further studies.

Former Conclusion:

In conclusion: Apoptosis of muscle fibre nuclei is present in inflammatory myopathies. The affected fibres express MHC I and are surrounded by CD8+ T-cells, macrophages and granzyme B+ cells. Fibres with apoptotic nuclei occur almost exclusively in myositis associated with partial invasion, and these processes occur in the same area, but rarely in the same fibre. The findings collectively support that apoptosis, induced by cytotoxic CD8+ T-cells in IIM, may be a mechanism activated in parallel with the fibre disintegration seen in partial invasion. In addition to its apoptosis inducing potential, granzyme B may have other important roles in IIM with partial invasion. Inflammatory cells with macrophage/myeloic dendritic properties are also present in infiltrates invading muscle fibres and adhere to fibres with apoptotic myonuclei.

New Conclusion:

In conclusion: Apoptosis of muscle fibre nuclei is present in inflammatory myopathies with partial invasions, and these processes occur in the same area, but rarely in the same fibre. The affected fibres express MHC I and are surrounded by CD8+ T-cells, macrophages and granzyme B+ cells. The findings collectively support that apoptosis, induced by cytotoxic CD8+ T-cells in IIM, may be a mechanism activated in parallel with the fibre disintegration seen in partial invasion. In addition to its apoptosis inducing potential, granzyme B may have other important roles in IIM with partial invasion. Inflammatory cells with macrophage/myeloic dendritic properties are also present in infiltrates invading muscle fibres and adhere to fibres with apoptotic myonuclei.

These text changes are red in Manuscript with Track Changes; those from the first revision are blue.

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