Immune checkpoint inhibitor therapy increases systemic SDF-1, cardiac DAMPs Fibronectin-EDA, S100/Calgranulin, galectine-3, and NLRP3-MyD88-chemokine pathways

Abstract

BackgroundImmune checkpoint inhibitors (ICIs) have significantly changed the oncology clinic in recent years, improving survival expectations in cancer patients. ICI therapy have a broad spectrum of side effects from endocrinopathies to cardiovascular diseases. In this study, pro-inflammatory and pro-fibrotic effects of short-term ICIs therapy in preclinical models were analyzed.MethodsFirstly, in a human in vitro model, human cardiomyocytes co-cultured with hPBMC were exposed to ICIs (with CTLA-4 or PD-1 blocking agents, at 200 nM) for 72 h. After treatment, production of DAMPs and 12 cytokines were analyzed in the supernatant through colorimetric and enzymatic assays. C57/Bl6 mice were treated with CTLA-4 or PD-1 blocking agents (15 mg/kg) for 10 days. Before (T0), after three days (T3) and after treatments (T10), ejection fraction, fractional shortening, radial and longitudinal strain were calculated by using bidimensional echocardiography (Vevo 2100, Fujfilm). Fibrosis, necrosis, hypertrophy and vascular NF-kB expression were analyzed through Immunohistochemistry. Myocardial expression of DAMPs (S100- Calgranulin, Fibronectin and Galectine-3), MyD88, NLRP3 and twelve cytokines have been analyzed. Systemic levels of SDF-1, IL-1β, and IL-6 were analyzed before, during and after ICIs therapy.ResultsRadial and longitudinal strain were decreased after 10 days of ICIs therapy. Histological analysis of NF-kB expression shows that short-term anti-CTLA-4 or anti-PD-1 treatment increased vascular and myocardial inflammation. No myocardial hypertrophy was seen with the exception of the pembrolizumab group. Myocardial fibrosis and expression of galectin-3, pro-collagen 1-α and MMP-9 were increased after treatment with all ICIs. Both anti-CTLA-4 or anti-PD-1 treatments increased the expression of DAMPs, NLRP3 inflammasome and MyD88 and induced both in vitro and in vivo the secretion of IL-1β, TNF-α and IL-6. Systemic levels of SDF-1, IL-1β and IL-6 were increased during and after treatment with ICIs.ConclusionsShort therapy with PD-1 and CTLA-4 blocking agents increases vascular expression of NF-kB, systemic SDF-1, IL-1β, IL-6 levels and myocardial NLRP3, MyD88 and DAMPs expression in preclinical models. A pro-inflammatory cytokine storm was induced in myocardial tissues and in cultured cardiac cells after ICIs therapy. The overall picture of the study suggests new putative biomarkers of ICIs-mediated systemic and myocardial damages potentially useful in clinical cardioncology.

Drug therapy for myocarditis induced by immune checkpoint inhibitors

“…In a preclinical mouse model, CTLA-4 knockout induced T cell and macrophage infiltration in myocardial tissues that destroyed cardiomyocytes, leading to metabolic failure with high mortality ( Wei et al, 2021 ). The activation of T cells and macrophages induces a proinflammatory phenotype in cardiac and vascular tissues by increasing the expression of nuclear factor-kappa B (NF-κB), the NLRP3 inflammasome, and MyD88 ( Quagliariello et al, 2022 ). The blockade of CTLA-4 in cardiomyocytes also led to an increased levels of proinflammatory cytokines, including those of interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), interleukin (IL)-2, and IL-17A in the myocardium ( Han et al, 2012 ).…”

“…A preclinical study showed that the depletion of T cells enhances silica-induced fibrosis by increasing the secretion of transforming growth factor-beta 1 (TGF-β1), which is associated with the suppression of CTLA-4 expression ( Liu et al, 2010 ). CTLA-4 blocking agents also increase myocardial fibrosis by modulating the expression of galectin-3, collagen 1, and matrix metalloproteinase 9 (MMP-9) ( Quagliariello et al, 2022 ). By contrast, a recent study demonstrated that CTLA4-Ig-mediated immunosuppression limits subepithelial fibrosis ( Khan et al, 2022 ).…”

“…TGF-β1 is a primary factor that drives fibrosis via the activation of Smad- and non-Smad-based signaling pathways that produce myofibroblasts ( Meng et al, 2016 ). PD-1 inhibitors also increase the expression of fibronectin, a major pro-fibrotic cytokine, in cardiomyocytes; fibronectin plays an important role in the fibrotic process ( Quagliariello et al, 2022 ).…”

Sodium-glucose cotransporter 2 inhibitor Dapagliflozin prevents ejection fraction reduction, reduces myocardial and renal NF-κB expression and systemic pro-inflammatory biomarkers in models of short-term doxorubicin cardiotoxicity

“…After treatments, hearts were weighed and cut lengthwise into two equal parts: half for immunohistochemistry (IHC) analysis and half for NLRP-3 and Myd-88 tissue quanti cation. Tissues were snap-frozen in dry ice until tissue homogenization, performed in a proper lysis buffer (0.1 M PBS, pH 7.4 + 1% Triton X-100 + protease inhibitor cocktail) and processed using a high intensity ultrasonic liquid processor [33]. The homogenates were centrifuged at 4 o C and supernatants were used to NLRP-3 and Myd-88 analysis through NLRP3 (Mouse NLRP3 ELISA Kit, OKEH05486, Aviva Systems Biology) and MyD88 (Mouse MyD88 ELISA Kit, OKEH03397 Aviva Systems Biology ).…”

Electrocardiographic characteristics of bladder cancer patients receiving preoperative chemotherapy combined with immunotherapy

“…On the other hand, ECG abnormalities in patients with ICI-associated myocarditis have been well described, with cardiac inflammation and fibrosis playing an important role(Zlotoff et al, 2021), but the exact mechanism of ECG alteration in asymptomatic patients treated with short-term ICI is currently unknown. In a preclinical study, Vincenzo et al found that 10 days of ICI treatment could increase myocardial fibrosis and induce pro-inflammatory cytokine storm through NLRP-3 and MyD-88 pathways(Quagliariello et al, 2022). Another basic research demonstrated that short-term ICI therapy in mice induces T-cell-mediated plaque inflammation and worsens plaque progression(Poels et al, 2020).…”