Cell death, it always matters

Abstract

We have read with great interest the article by Halili et al. [1], in which the authors propose that TSA suppresses the LPSinduced mRNA expression of the proinflammatory mediators endothelin-1, Ccl-7/MCP-3, and IL-12p40 but amplifies the expression of the proatherogenic factors Cox-2 and Pai-1 in mouse BMM.We totally agree with the initial reflections made by the authors relating the complexity of the effects exerted by HDAC inhibitors, not only in macrophages but also in other cell types, and the existence of even contradictory effects, some of them anti-inflammatory or antiproliferative, although others are clearly proinflammatory. However, we find very difficult to appreciate or support how the authors reached those reflections regarding the value or involvement of apoptosis on the observed effects of TSA.The differences observed in the effects of HDAC inhibitors based on a gradient of drug concentration in macrophages could be easily related to the fact that these HDAC inhibitors are not selective. Moreover, we and others [2,3] have seen previously that the sensitivity of HDAC inhibitors also depends on several factors, including the differentiation state, confluence, or proliferative rate of different cell types. Similarly to the authors, we have also addressed the relation between macrophage proliferation/viability/activation and HDAC effects [3]. In this regard, our previous data suggest that in those cells in which the activation stimuli do not affect proliferation, such as intestinal epithelial cells, or even induce proliferation, such as lymphocytes, butyrate (a pan-HDAC inhibitor) inhibits activation almost totally through the induction of apoptosis; in contrast, in those cells, such as bone marrow macrophages, whose activation induces a blockage of the proliferation, butyrate is unable to inhibit their activation and slightly affects cellular survival. Inasmuch, even in the same cellular type, such as the case of macrophages, LPS-activated RAW 264.7 (as a proliferating cell line) and LPS-activated BMM (quiescent primary cultures) present different proinflammatory responses [3].However, these studies are always made at physiological butyrate concentrations, and it is therefore difficult for us to understand the rationale behind the nonpharmacological range of concentrations selected by the authors to test the effects of the HDAC inhibitors [1], as they are more than one log higher than the dose required to inhibit HDAC. Moreover, we do not understand the conclusions drawn by the authors that the high degree of cell death induced by these concentrations is not relevant or involved in the observed effects.Cell death at the degrees observed by the authors (Ͻ20% of viability after stimuli) should always be taken in consideration. Although the authors clearly observed that the Cox-2-and Pai-1-induced expression is observed only at these concentrations and with those inhibitors that clearly affect cell viability [1], they conclude that the induction of apoptosis is not responsible for this observed effect, ...

Dead cells certainly do matter, particularly when they can speak from the grave

“…A much simpler explanation, however, is that different inflammatory mediators were examined. We showed that noncytotoxic TSA concentrations inhibited LPS-inducible mRNA expression of IL12p40, endothelin 1, and Ccl7 (qPCR), as well as IL-12p40 protein secretion (ELISA) in BMM, and Comalada et al [2] showed that noncytotoxic butyrate concentrations did not affect LPS-inducible mRNA expression of iNOS and Cox-2 (semiqPCR) or TNF secretion (ELISA) in the same cells. As HDAC inhibitors do not target classical TLR signaling pathways (e.g., MAPKs, NF-B) and suppress only a subset of TLR4-dependent, inflammatory responses, it is not surprising that there were differences between the two studies; different pharmacological reagents were used to examine different inflammatory outputs.…”

“…Comalada and colleagues [2] suggest that these data are meaningless, as the cells eventually die. They argue that the amplifying effects of HDAC inhibitors on PAI-1 protein levels relate to phagocytosis and HDAC inhibitor-induced cell death.…”

“…We therefore determined whether differential effects of broad-spectrum HDAC inhibitors on TLR4 responses in macrophages were separable [1]. Comalada and colleagues [2] suggest that HDAC inhibitors do not exert anti-inflammatory effects at noncytotoxic concentrations when used on cells where the activation stimulus inhibits cell proliferation (e.g., LPS-activated primary mouse macrophages). They also argue that our studies are not biologically relevant because of the concentrations of HDAC inhibitors used.…”

Crippling life support for SARS-CoV-2 and other viruses through synthetic lethality

“…SARS-CoV-2 nsp5 interacts with histone deacetylase 2 and tRNA methyltransferase 1, which have putative nsp5-specific cleavage sites (Gordon et al, 2020a). Degradation of histone deacetylase 2 might suppress host inflammatory and immune signaling responses (Comalada et al, 2010;Guise et al, 2013;Roger et al, 2011), while loss of tRNA methyltransferase 1 may impair host-protein translation and redox homeostasis (Dewe et al, 2017). Loss of either protein would sensitize SARS-CoV-2-infected cells to drugs targeting their synthetic lethal partners.…”