Cristina Chimenti1,2, Romina Verardo2, Fernanda Scopelliti3, Claudia Grande1, Nicola Petrosillo4, Pierluca Piselli5, Ruggero De Paulis6, Andrea Frustaci1,2. 1. Cardiovascular, Respiratory, Nephrologic, Anesthesiologic and Geriatric Sciences, La Sapienza University, Rome, Italy. 2. Cellular and Molecular Cardiology Laboratory, IRCCS INMI L. Spallanzani, Rome, Italy. 3. 'Bambino Gesù' Children's Hospital, Rome, Italy. 4. Clinical and Research Department, IRCCS INMI L. Spallanzani, Rome, Italy. 5. Unit of Clinical Epidemiology, IRCCS INMI L. Spallanzani, Rome, Italy. 6. Department of Cardiac Surgery, European Hospital, Rome, Italy.
Abstract
AIMS: We sought to determine whether myocardial expression of Toll-like receptor 4 (TLR4) may predict the response to immunosuppression. METHODS AND RESULTS: Endomyocardial biopsies from 237 patients with virus-negative inflammatory cardiomyopathy treated with immunosuppression were retrospectively examined for the expression of TLR4, differentiating those patients responding to immunosuppression (n = 193) from non-responder patients (n = 44). A semiquantitative evaluation of the immunoreactivity (grading from 0 to 4) for TLR4 and human leucocyte antigen (HLA)-DR was performed together with real-time PCR and western blot for TLR4. Cardiomyocyte apoptosis and necrosis was evaluated by in situ ligation with hairpin probes. A focal intense positive cytoplasmic immunostaining for TLR4 was observed in cardiomyocytes of all responders (P < 0.001 vs. non-responders). A grading 2 or above (2+) at baseline showed a sensitivity of 100% and 90.9% specificity with a positive predictive value of 98% as a predictor of an immunosuppression-positive response. Real-time PCR and western blot analysis for TLR4 were 4.3-fold and 4.6-fold higher, respectively, in responders vs. non-responders. Correlation between TLR4 grading and TLR4 mRNA molecular and protein expression was highly significant. HLA-DR did not discriminate between the two groups. Cardiomyocyte death by apoptosis was 3.7-fold higher in responders vs. non-responders and significantly correlated with TLR4 expression, while necrosis was comparable. Intensity of baseline TLR4 expression correlated with the variation in ejection fraction after 6 months of immunosuppression. CONCLUSION: TLR4 is highly expressed in human myocarditis responding to immunosuppression. It can be considered as a new sensitive marker in patient selection predicting a good response to immunosuppressive therapy.
AIMS: We sought to determine whether myocardial expression of Toll-like receptor 4 (TLR4) may predict the response to immunosuppression. METHODS AND RESULTS: Endomyocardial biopsies from 237 patients with virus-negative inflammatory cardiomyopathy treated with immunosuppression were retrospectively examined for the expression of TLR4, differentiating those patients responding to immunosuppression (n = 193) from non-responder patients (n = 44). A semiquantitative evaluation of the immunoreactivity (grading from 0 to 4) for TLR4 and human leucocyte antigen (HLA)-DR was performed together with real-time PCR and western blot for TLR4. Cardiomyocyte apoptosis and necrosis was evaluated by in situ ligation with hairpin probes. A focal intense positive cytoplasmic immunostaining for TLR4 was observed in cardiomyocytes of all responders (P < 0.001 vs. non-responders). A grading 2 or above (2+) at baseline showed a sensitivity of 100% and 90.9% specificity with a positive predictive value of 98% as a predictor of an immunosuppression-positive response. Real-time PCR and western blot analysis for TLR4 were 4.3-fold and 4.6-fold higher, respectively, in responders vs. non-responders. Correlation between TLR4 grading and TLR4 mRNA molecular and protein expression was highly significant. HLA-DR did not discriminate between the two groups. Cardiomyocyte death by apoptosis was 3.7-fold higher in responders vs. non-responders and significantly correlated with TLR4 expression, while necrosis was comparable. Intensity of baseline TLR4 expression correlated with the variation in ejection fraction after 6 months of immunosuppression. CONCLUSION:TLR4 is highly expressed in humanmyocarditis responding to immunosuppression. It can be considered as a new sensitive marker in patient selection predicting a good response to immunosuppressive therapy.