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mTORC1 activation associated with granuloma formation in mice.
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Potential clinical biomarker and therapeutic target for sarcoidosis.
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High expression of mTOR and downstream effectors in sarcoid granulomas.
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mTOR activation neither related to disease severity nor the need for therapy.
Abstract
Introduction
Sarcoidosis is a systemic granulomatous disease with a variable clinical presentation and disease course. There is still no reliable biomarker available, which assists in the diagnosis or prediction of the clinical course. According to a murine model, the expression level of the metabolic checkpoint kinase mechanistic target of Rapamycin complex 1 (mTORC1) in granulomas of sarcoidosis patients may be used as a clinical biomarker.
Material and methods
This is a retrospective analysis of 58 patients with histologically confirmed sarcoidosis. Immunohistochemical staining of granulomas from tissue samples was evaluated for the expression of activated mTORC1 signaling, including phosphorylated mTOR, its downstream effectors S6K1, 4EBP1 and the proliferation marker Ki-67. Patients were categorized according to different clinical phenotypes, serum biomarkers, and immunomodulatory therapy.
Results
All patients showed activated mTORC1 signaling in granulomas, which correlated with its downstream effectors S6K1 and 4EBP1 but was not related to Ki-67 expression. The mTORC1 activity revealed an association neither to disease severity nor the necessity of treatment; however, p-mTOR inversely correlated with cumulative corticosteroid dosage.
Conclusion
Our data confirm activation of the mTORC1 pathway in sarcoidosis, supporting the hypothesis that mTOR is a significant driver in granuloma formation. However, we could not find a relationship between the degree of mTOR activation and disease severity or the need for therapy.
Sarcoidosis is a heterogeneous, systemic disease characterized by non-caseating granulomas mainly affecting lymphatic nodes, the lung, skin, and liver [
]. Genetic susceptibility, as well as specific environmental conditions, have been suggested to contribute to disease onset, and granuloma formation is considered a hallmark in pathogenesis [
]. Morphological characteristics of granulomas have been widely investigated, but the exact molecular pathway resulting in macrophage aggregation has not yet been identified [
] forms the catalytic subunit of two protein complexes called mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) and plays a pivotal role in cell physiology regulating the function and metabolism of innate and adaptive immune cell populations [
mTORC1 regulates protein synthesis through phosphorylation of two main target proteins: the S6 kinase 1 (S6K1) and the eukaryotic initiation factor 4E-binding protein (4EBP1) [
Linke et al. were the first to investigate the role of the metabolic checkpoint kinase mTORC1 in granuloma formation using a tuberous sclerosis protein 2 (Tsc2)-knockout murine model, resulting in the spontaneous formation of granulomas [
]. mTORC1 inhibited apoptosis and induced macrophage proliferation via induction of cyclin-dependent kinase 4 (CDK4) expression and metabolic reprogramming [
They further identified a high number of Ki-67 positive macrophages in the granulomas. Ki-67 is a DNA-binding protein expressed by proliferating cells [
]. The Ki-67 proliferative index, i.e., the identification of the percentage of Ki-67 positive cells by immunohistochemistry (IHC) is widely used to quantify the growth fraction of tumor cells [
A well-established clinical biomarker to monitor disease activity in sarcoidosis is lacking; thus, associations of the described activated mTOR signaling to disease activity are of major significance. In a small cohort of patients with sarcoidosis, its activation was associated with disease progression [
]. mTORC1 signaling involvement has further been identified in RNA-seq gene set enrichment data from a cutaneous sarcoidosis patient and successful treatment of a sarcoidosis patient with the mTOR inhibitor rapamycin has been reported ([
Considering this evidence, we hypothesized a relationship between mTOR signaling intensity and prognosis of patients with sarcoidosis. Therefore, we analyzed the IHC expression of phosphorylated mTOR (p-mTOR) as well as its two downstream markers p-S6K1, and p-4EBP1. We further investigated a possible association of p-mTOR activation with clinical outcome, especially the need for higher corticosteroid dosing or the need for a second immunosuppressant and disease relapses rate.
2. Material and methods
2.1 Subjects and ethics
This is a cross-sectional single-center study, including 58 patients with a histologically verified sarcoidosis. Patients were retrospectively recruited from the sarcoidosis registry of the pulmonary outpatient clinic of the Medical University of Innsbruck and included if patients had at least a follow up of two years, the disease was histologically confirmed and a specimen for further IHC testing still available. Biopsy specimens were collected to confirm the diagnosis and patients were therapy-naïve at this time point. Baseline characteristics (age, gender, body mass index (BMI) and co-morbidities as reported in the electronic patient record), inflammatory biomarkers (C-reactive protein (CRP) and angiotensin-converting enzyme (ACE)), sarcoidosis-therapy (defined as an oral corticosteroid (OCS), additional immunosuppressant therapy (AIS) including methotrexate (MTX), mycofenolatmofetil (MMF), azathioprine (AZA)) as well as organ manifestation were recorded. The mean follow-up observation time was 86.1 ± 49.46 months.
The course of the disease was classified according to the four chest-radiographic stages by Scadding et al. [
], which categorizes patients into six stages: (1) acute onset, no need for immunosuppressive therapy, (2) acute onset, one period of treatment, not lasting longer than one year, (3) acute onset, need for several periods of immunosuppressive therapy or long-lasting treatment (>12 months), (4) subacute onset, no need for immunosuppressive therapy, (5) subacute onset, one period of immunosuppressive treatment, not lasting longer than one year and (6) subacute onset, need for several periods of immunosuppressive treatment or long-lasting treatment (>12 months).
All procedures performed in the present study involving human participants were in accordance with the ethical standards of the Institutional and/or National Research Committee and with the 1964 Helsinki declaration and its later amendments and were performed after approval of the Ethics Committee of the Medical University of Innsbruck (approval numbers: AN2015-0069 347/4.14 372/5.10 (4048a)).
2.2 Immunohistochemistry
Granuloma specimens from lung (5 samples), peripheral lymph nodes (9 samples), mediastinal lymphnodes (34 samples) and skin (9 samples) have been stained with the following antibodies: p-mTOR, p-S6K1, p-4EBP1, and the cellular marker for proliferation Ki-67 (Fig. 1).
Fig. 1Example of different antibody expressions in sarcoid granulomas. (a) Haematoxylin-eosin staining, (b) p-mTOR, (c) p-S6K1, (d) p-4EBP1.
The IHC-score used for p-mTOR, p-4EBP1, and p-S6K1 contains two separate scores (Table 1). The first score includes the percentage of antibody-positive cells in the granulomas with 0 indicating <10%, 1 indicating 10–25%, 2 indicating 25–50%, 3 indicating 50–75%, and 4 indicating >75%. The second score describes staining intensity ranging from 0 to 3. The addition of the score of antibody positive cells and staining intensity depicted in Table 1 results in a summative IHC-score ranging from 0 to 7.
Table 1Immunohistochemistry score classification for p-mTOR, p-4EBP1 and p-S6K1. Addition of the scores for the percentage of antibody positive cells (0-4) and staining intensity (0-3) results in the overall/summative immunohistochemistry score.
The IHC scoring for Ki-67 ranged between 0 and 2. 0 equals <1% expression, 1 equals 1–5% expression and 2 equals >5% expression.
2.3 Statistical analyses
Mean comparison of normally distributed numeric data was performed using Student's t-test. If Gaussian distribution was not given, the Mann-Whitney-U-test and Kruskal-Wallis-test were applied. Baseline characteristics in terms of categorical variables were compared using Chi-Square, and Fisher's exact test, where appropriate. Spearman rank correlation technique was used for the analysis of monotonic associations in non-normally distributed data. If Gaussian distribution based on Shapiro-Wilk test and a linear relationship were given, Pearson correlation coefficient was calculated to assess the degree of correlation. All tests were two-sided, and a p-value of 0.05 indicated statistical significance. Statistical analyses were performed with the SPSS 24.0 statistical package (IBM Corp., Armonk, NY, USA).
3. Results
The mean age of the 58 included patients was 54.52 ± 12.63 years and 32 patients (55.2%) were male. The baseline parameters of the cohort are shown in Table 2.
Table 2Baseline characteristics of the study cohort. Quantitative parameters are represented as mean ± standard deviation. Categorical parameters are represented as total n and percentage. BMI = Body-mass-index; OCS = oral corticosteroid therapy; AIS = additional immunosuppressant therapy; SCAC = Sarcoid Clinical Activity Classification; CRP = C-reactive protein; ACE = angiotensin converting enzyme; ref.range = reference range.
The most frequently involved organs were hilar lymph-nodes (n = 55, 94.8%), followed by the lung (n = 47, 81%) and skin (n = 40, 40%). Detailed information about organ involvement is given in Fig. 2. In total, 48 patients (82.8%) presented with extrapulmonary manifestation.
Fig. 2Chart of the various organ manifestations. Data is given as a relative number (%).
According to the defined classification systems, Löfgren syndrome was present in 9 patients (15.5%) and according to Scadding radiographic classification, stage 3 was the most frequent stage at first presentation. During follow-up, half of the patients (50%) were down-staged, while the remaining 29 patients had the same radiographic staging or were upstaged. By using the SCAC classification, stage 6 was the most frequent (n = 18, 34%), followed by stage 4 (n = 12, 22.6%) and stage 3 (n = 10, 18.9%). Further disease classification details are given in Table 2.
IHC analyses were performed in all 58 histologic samples and the results are summarized in Table 3.
Table 3Immunhistochemic scoring of histologic samples for p-mTOR and the downstream markers of activated mTORC1 signaling p-S6K1 and p-4EBP1 ranging from 0 to 7, as well as the cellular proliferation marker Ki-67 ranging from 0 to 2.
The mean p-mTOR score was 4.5 ± 1.9, the mean p-S6K1 score was 4.8 ± 2.0, the mean p-4EBP1 score was 6.2 ± 1.1, and none of the analyzed samples scored null for either one of the tested targets. The majority of patients had a Ki-67 score of 1 (n = 33, 56.9%) and 17 patients (29.3%) scored 2, while 8 (13.8%) patients scored 0. p-mTOR scores correlated with p-4EBP1 scores (r = 0.27, p = 0.04) and p-S6K1 scores (r = 0.344, p < 0.01). In contrast, no significant associations could be detected with Ki-67 (r = 0.21, p > 0.1). However, Ki-67 correlated with p-S6K1 (r = 0.344, p < 0.01). Further, a significant correlation was found between p-4EBP1 and p-S6K1 expression levels (r = 0.471, p < 0.01). Neither serum CRP nor ACE did significantly correlate with p-mTOR, p-S6K1, p-4EBP1 or Ki-67.
When comparing patients with and without Löfgren-syndrome, no significant differences in the scores of p-mTOR, p-S6K1, p-4EBP1 and Ki-67 were observed (p > 0.1). Similar findings could be described, when comparing p-mTOR, p-S6K1, p-4EBP1 and Ki-67 scores in patients with only pulmonary or with extrapulmonary manifestation, when comparing the four radiographic stages according to Scadding (p > 0.1). The same applied when comparing subjects anticipating an improvement of sarcoidosis as compared to those with stability or worsening of radiographic staging, according to Scadding (p > 0.1). Also, no significant differences of p-mTOR, p-S6K1, p-4EBP1, and Ki-67 scores were observed between the various SCAC stages (p > 0.1), and no differences were found between patients with and without therapy or those with or without the need of an AIS, respectively. The detailed comparisons of p-mTOR, p-S6K1, p-4EBP1, and Ki-67 scores according to various organ involvements are presented in Table 4. The scores of p-S6K1, p-4EBP1, and Ki-67 did not significantly correlate with the cumulative dosages of oral corticosteroids (OCS; referred to as per kg bodyweight); however, p-mTOR expression inversely correlated with cumulative OCS (r = −0-336, p = 0.02).
Table 4Comparison of p-mTOR, p-S6K1, p-4EBP1 and Ki-67 scores according to various organ involvements. Grey boxes indicate no significant difference (p > 0.1), red boxes indicate significantly elevated scores (p < 0.05), and green boxes indicate a trend (p < 0.1) towards elevated scores.
Table 4Comparison of p-mTOR, p-S6K1, p-4EBP1 and Ki-67 scores according to various organ involvements. Grey boxes indicate no significant difference (p > 0.1), red boxes indicate significantly elevated scores (p < 0.05), and green boxes indicate a trend (p < 0.1) towards elevated scores.
4. Discussion
Rising morbidity and mortality rates of patients with sarcoidosis require a better understanding of different molecular phenotypes of the disease and the development of targeted anti-inflammatory therapies [
]. Previously reported data describing mTORC1 activation to result in granuloma formation in mice, as well as of mTORC1 signaling activation in a small cohort of 15 patients being associated with progressive disease, stimulated the hypothesis of sarcoidosis activity being related to the intensity of mTORC1 signaling [
This study revealed active mTORC1 signaling in all patients of a real-life sarcoidosis cohort independent of its clinical presentation. To quantify the expression of mTORC1 in sarcoid granulomas its catalytic core p-mTOR, as well as its downstream regulators p-S6K1 and p-4EBP1, have been determined by IHC. Results have been interpreted with a scoring system consisting of the percentage of antibody-positive cells and staining intensity, referring to scoring systems used to measure mTOR expression in oncological settings [
]. Patients of our cohort not only showed high scores of p-mTOR but also of its phosphorylated downstream metabolites that were associated with p-mTOR expression. In contrast, Linke et al. described active mTORC1 signaling in granulomatous lesions only in 33% out of 27 biopsies in an initial screening. The reason for the different signaling intensity in comparison to our data is at this stage elusive. However, differences in clinical sarcoidosis presentation, cohort size and diverging definitions of IHC stains are possible confounders.
The absence of an association between p-mTOR and Ki-67 expression was admittedly surprising. However, mTOR signaling is involved in numerous cellular processes and proliferation only comprises one facet [
]. The relation of Ki-67 to the expression of its downstream effector p-S6K1 expression matches the presence of higher p-S6K1 levels in the high Ki-67 expression group in the study by Linke et al., emphasizing the significance of macrophage proliferation in sarcoidosis [
Characterization of mTOR expression is of significant interest as there are still no established biomarkers for evaluating clinical courses and the necessity of treatment in sarcoidosis. Frequently used parameters to estimate clinical course like ACE and soluble interleukin-2 receptor (s-IL2R) are unreliable. Although it has been shown that ACE has a high negative predictive value for detecting sarcoidosis in undifferentiated uveitis [
], previous investigations found a reduced sensitivity and insufficient specificity of ACE comparing high values of the average population with sarcoidosis patients in a large population-based study [
Serum soluble interleukin-2 receptor level is more sensitive than angiotensin-converting enzyme or lysozyme for diagnosis of sarcoidosis and may be a marker of multiple organ involvement.
] support the pathophysiologic role of mTORC1 in sarcoidosis. Moreover, mTOR pathways potentially offer a specific therapeutic target; therefore, its assessment could act as a novel theragnostic biomarker. Regarding its functional relevance, the observed activation of mTORC1 signaling suggests a disrupted autophagy process. Pacheco et al. suggested that genetic mutations related to sarcoidosis mostly affect autophagy genes and the regulatory hubs mTOR and Rac1 emphasizing their role in sarcoidosis pathogenesis [
Our data are confirmative in showing that the mTORC1 pathway is highly active in sarcoid granulomas with activated p-mTOR and its downstream targets p-S6K1and 4EBP1. However, mTORC1 is activated in biopsy specimens of all patients with sarcoidosis and its expression levels are not linked to the clinical severity, the subsequent course or the therapeutic needs. Thus, unspecific staining of p-mTOR has to be discussed. Yet, IHC analysis of p-mTOR expression in other diseases was not positive for all the samples evaluated, hence unspecific staining, in this case, seems unlikely [
]. No significant differences in p-mTOR, p-S6K1, p-4EBP1, and Ki-67 expression could be detected when comparing patients with or without Löfgren's syndrome, or between the different clinical stages neither when applying the SCAC nor the Scadding classification system. Furthermore, the expression of p-mTOR, p-S6K1, p-4EBP1 and Ki-67 were not associated with the cumulative amount of OCS; nevertheless, an inverse relationship between p-mTOR and OCS could be detected. The latter was somewhat surprising as we would have expected a high expression of p-mTOR to be associated with a more progressive disease and therefore, to require more OCS. The outcomes of the here presented study can thus not confirm the findings of Linke et al., who showed an increased mTORC1 activity in patients with progressive disease. The association of a higher p-mTOR expression with the need for less OCS stands in complete contradiction to our hypotheses and needs clarification in future studies.
Although the here presented data show promising new aspects of mTORC1 signaling in sarcoidosis patients, we have to acknowledge several limitations. The major limitation is the retrospective study design. Biopsy specimens of sarcoid granulomas already stored at the Department of Pathology had to be re-stained and were subsequently re-analyzed by IHC. IHC analysis only allows a snapshot of the complex regulation of mTORC1 activity in our sarcoidosis cohort. Moreover, our cohort showed a distinct amount of severe cases requiring long term immunosuppressive treatment in comparison to published literature [
], most likely due to selection bias (center of referral for severe cases) or by the size of the cohort. Therefore, further prospective studies, including approaches in RNomics/proteomics in a large prospective cohort, are warranted to clarify the significance of mTOR expression and the role of its downstream regulators.
5. Conclusion
In summary, we report activation of the mTOR pathway in all biopsy specimens from sarcoidosis patients, indicating activation of mTORC1 in any granuloma. However, the degree of mTOR activation was not linked to disease severity nor the necessity of treatment. Since mTOR pathways offer a specific therapeutic target, further studies phenotyping sarcoidosis patients in order to evaluate targeted therapies are highly awaited.
CRediT authorship contribution statement
Alex Pizzini: Writing - original draft, Conceptualization, Funding acquisition, Formal analysis, Data curation, wrote the manuscript, contributed to the conception and design of the study, the acquisition of data and the analysis and interpretation. Hannes Bacher: Conceptualization, Funding acquisition, Formal analysis, Data curation, Writing - original draft, contributed to the conception and design of the study, the acquisition of data and the analysis and interpretation and co-wrote the paper. Magdalena Aichner: Conceptualization, Formal analysis, Data curation, Writing - original draft, co-wrote the paper, contributed to the conception and design of the study, the data analysis and interpretation. Alexander Franchi: Funding acquisition, Data curation, study design, data acquisition and interpretation. Kathrin Watzinger: Formal analysis, Data curation, study design, data acquisition and interpretation. Ivan Tancevski: Conceptualization, conception and design of the study. Thomas Sonnweber: Formal analysis, Data curation, study design, data analysis and interpretation. Birgit Mosheimer-Feistritzer: Formal analysis, Data curation, study design, data analysis and interpretation. Christina Duftner: Formal analysis, Data curation, study design, data analysis and interpretation. Bettina Zelger: Funding acquisition, Data curation, contributed to the design of the study design, the acquisition of data and interpretation. Johannes Pallua: Formal analysis, Data curation, study design, data analysis and interpretation. Susanne Sprung: Funding acquisition, Data curation, study design, data analysis and interpretation. Thomas Weichhart: Conceptualization, conception and design of the study. Bernhard Zelger: Funding acquisition, Formal analysis, Data curation, study design, the acquisition of data, data analysis and interpretation. Günter Weiss: Conceptualization, Formal analysis, Data curation, conception and design of the study, data analysis and interpretation. Judith Löffler-Ragg: Conceptualization, Formal analysis, Data curation, conception and design of the study, data analysis and interpretation.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Serum soluble interleukin-2 receptor level is more sensitive than angiotensin-converting enzyme or lysozyme for diagnosis of sarcoidosis and may be a marker of multiple organ involvement.
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