Therapeutic reconditioning of damaged lungs by transient heat stress during ex vivo lung perfusion.

Ex vivo lung perfusion (EVLP) may serve as a platform for the pharmacologic repair of lung grafts before transplantation (LTx). We hypothesized that EVLP could also permit nonpharmacologic repair through the induction of a heat shock response, which confers stress adaptation via the expression of heat shock proteins (HSPs). Therefore, we evaluated whether transient heat application during EVLP (thermal preconditioning [TP]) might recondition damaged lungs before LTx. TP was performed during EVLP (3 hours) of rat lungs damaged by warm ischemia by transiently heating (30 minutes, 41.5 °C) the EVLP perfusate, followed by LTx (2 hours) reperfusion. We also assessed the TP (30 minutes, 42 °C) during EVLP (4 hours) of swine lungs damaged by prolonged cold ischemia. In rat lungs, TP induced HSP expression, reduced nuclear factor κB and inflammasome activity, oxidative stress, epithelial injury, inflammatory cytokines, necroptotic death signaling, and the expression of genes involved in innate immune and cell death pathways. After LTx, heated lungs displayed reduced inflammation, edema, histologic damage, improved compliance, and unchanged oxygenation. In pig lungs, TP induced HSP expression, reduced oxidative stress, inflammation, epithelial damage, vascular resistance, and ameliorated compliance. Collectively, these data indicate that transient heat application during EVLP promotes significant reconditioning of damaged lungs and improves their outcomes after transplantation.

Experimental Models of Ischemic Lung Damage for the Study of Therapeutic Reconditioning During Ex Vivo Lung Perfusion.

Background: Ex vivo lung perfusion (EVLP) may allow therapeutic reconditioning of damaged lung grafts before transplantation. This study aimed to develop relevant rat models of lung damage to study EVLP therapeutic reconditioning for possible translational applications. Methods: Lungs from 31 rats were exposed to cold ischemia (CI) or warm ischemia (WI), inflated at various oxygen fractions (FiO2), followed by 3 h EVLP. Five groups were studied as follow: (1) C21 (control): 3 h CI (FiO2 0.21); (2) C50: 3 h CI (FiO2 0.5); (3) W21: 1 h WI, followed by 2 h CI (FiO2 0.21); (4) W50: 1 h WI, followed by 2 h CI (FiO2 0.5); and (5) W2h: 2 h WI, followed by 1 h CI (FiO2 0.21). Following 3 h EVLP, we measured static pulmonary compliance (SPC), pulmonary vascular resistance, lung weight gain (edema), oxygenation capacity (differential partial pressure of oxygen), and protein carbonyls in lung tissue (oxidative stress), as well as lactate dehydrogenase (LDH, lung injury), nitrotyrosine (nitro-oxidative stress), interleukin-6 (IL-6, inflammation), and proteins (permeability edema) in bronchoalveolar lavage (BAL). Perivascular edema was quantified by histology. Results: No significant alterations were noted in C21 and C50 groups. W21 and W50 groups had reduced SPC and disclosed increased weight gain, BAL proteins, nitrotyrosine, and LDH. These changes were more severe in the W50 group, which also displayed greater oxidative stress. In contrast, both W21 and W50 showed comparable perivascular edema and BAL IL-6. In comparison with the other WI groups, W2h showed major weight gain, perivascular edema, SPC reduction, drop of differential partial pressure of oxygen, and massive increases of BAL LDH and proteins but comparable increase of IL-6 and biomarkers of oxidative stress. Conclusions: These models of lung damage of increasing severity might be helpful to evaluate new strategies for EVLP therapeutic reconditioning. A model combining 1 h WI and inflation at FiO2 of 0.5 seems best suited for this purpose by reproducing major alterations of clinical lung ischemia-reperfusion injury.

Effects of cold or warm ischemia and ex-vivo lung perfusion on the release of damage associated molecular patterns and inflammatory cytokines in experimental lung transplantation.

BACKGROUND: Lung transplantation (LTx) is associated with sterile inflammation, possibly related to the release of damage associated molecular patterns (DAMPs) by injured allograft cells. We have measured cellular damage and the release of DAMPs and cytokines in an experimental model of LTx after cold or warm ischemia and examined the effect of pretreatment with ex-vivo lung perfusion (EVLP). METHODS: Rat lungs were exposed to cold ischemia alone (CI group) or with 3h EVLP (CI-E group), warm ischemia alone (WI group) or with 3 hour EVLP (WI-E group), followed by LTx (2 hour). Bronchoalveolar lavage (BAL) was performed before (right lung) or after (left lung) LTx to measure LDH (marker of cellular injury), the DAMPs HMGB1, IL-33, HSP-70 and S100A8, and the cytokines IL-1ß, IL-6, TNFα, and CXCL-1. Graft oxygenation capacity and static compliance after LTx were also determined. RESULTS: Compared to CI, WI displayed cellular damage and inflammation without any increase of DAMPs after ischemia alone, but with a significant increase of HMGB1 and functional impairment after LTx. EVLP promoted significant inflammation in both cold (CI-E) and warm (WI-E) groups, which was not associated with cell death or DAMP release at the end of EVLP, but with the release of S100A8 after LTx. EVLP reduced graft damage and dysfunction in warm ischemic, but not cold ischemic, lungs. CONCLUSIONS: The pathomechanisms of sterile lung inflammation during LTx are significantly dependent on the conditions. The release of HMGB1 (in the absence of EVLP) and S100A8 (following EVLP) may be important factors in the pathogenesis of LTx.

Treatment with 3-aminobenzamide during ex vivo lung perfusion of damaged rat lungs reduces graft injury and dysfunction after transplantation.

Ex vivo lung perfusion (EVLP) with pharmacological reconditioning may increase donor lung utilization for transplantation (LTx). 3-Aminobenzamide (3-AB), an inhibitor of poly(ADP-ribose) polymerase (PARP), reduces ex vivo lung injury in rat lungs damaged by warm ischemia (WI). Here we determined the effects of 3-AB reconditioning on graft outcome after LTx. Three groups of donor lungs were studied: Control (Ctrl): 1 hour WI + 3 hours cold ischemia (CI) + LTx; EVLP: 1 hour WI + 3 hours EVLP + LTx; EVLP + 3-AB: 1 hour WI + 3 hours EVLP + 3-AB (1 mg. mL-1 ) + LTx. Two hours after LTx, we determined lung graft compliance, edema, histology, neutrophil counts in bronchoalveolar lavage (BAL), mRNA levels of adhesion molecules within the graft, as well as concentrations of interleukin-6 and 10 (IL-6, IL-10) in BAL and plasma. 3-AB reconditioning during EVLP improved compliance and reduced lung edema, neutrophil infiltration, and the expression of adhesion molecules within the transplanted lungs. 3-AB also attenuated the IL-6/IL-10 ratio in BAL and plasma, supporting an improved balance between pro- and anti-inflammatory mediators. Thus, 3-AB reconditioning during EVLP of rat lung grafts damaged by WI markedly reduces inflammation, edema, and physiological deterioration after LTx, supporting the use of PARP inhibitors for the rehabilitation of damaged lungs during EVLP.

Murine Myocardial Infarction Model using Permanent Ligation of Left Anterior Descending Coronary Artery.

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine models of MI with permanent ligation of left-anterior descending (LAD) coronary artery closely mimics MI in humans. Murine models benefit from the extensive genetic engineering available nowadays. Here we propose a reproducible murine surgical model of myocardial infarction by permanent LAD coronary ligation. Our technique comprises anesthesia with ketamine/xylazine that can be rapidly reversed by administration of an antagonist, intubation without tracheotomy for mechanical-assisted ventilation, ventilation with application of extrinsic positive end-expiratory pressure (PEEP) to avoid alveolar collapse, a thoracotomy method limiting to the minimum surgical lesions made to skeletal muscles, and lung inflation without thoracentesis. This method is sparsely invasive, reproducible and reduces post-surgery mortality and complications.

The AIM2 inflammasome: Sensor of pathogens and cellular perturbations.

Recognition of pathogens and altered self must be efficient and highly specific to orchestrate appropriate responses while limiting excessive inflammation and autoimmune reaction to normal self. AIM2 is a member of innate immune sensors that detects the presence of DNA, arguably the most conserved molecules in living organisms. However, AIM2 achieves specificity by detecting altered or mislocalized DNA molecules. It can detect damaged DNA, and the aberrant presence of DNA within the cytosolic compartment such as genomic DNA released into the cytosol upon loss of nuclear envelope integrity. AIM2 is also a key sensor of pathogens that detects the presence of foreign DNA accumulating in the cytosol during the life cycle of intracellular pathogens including viruses, bacteria, and parasites. AIM2 activation initiates the assembly of the inflammasome, an innate immune complex that leads to the activation of inflammatory caspases. This triggers the maturation and secretion of the cytokines IL-1ß and IL-18. It can also initiate pyroptosis, a proinflammatory form of cell death. The AIM2 inflammasome contributes to physiological responses and diseases. It is a key player in host defenses, but its deregulation can contribute immune-linked diseases, such as autoinflammatory and autoimmune pathologies. Moreover, AIM2 may play a role in cancer development. Recent studies have shown that the detection of self-DNA species by AIM2 is an important factor that contributes to diseases associated with perturbation of cellular homeostasis. Thus, in addition of being a sensor of pathogen associated molecular patterns (PAMPs), the AIM2 inflammasome is emerging as a key guardian of cellular integrity.

Hydrogen sulfide inhibits NLRP3 inflammasome activation and reduces cytokine production both in vitro and in a mouse model of inflammation.

A variety of stimuli, including monosodium urate (MSU) crystals, activate the NLRP3 inflammasome, and this activation involves several molecular mechanisms including xanthine oxidase (XO) up-regulation and mitochondrial dysfunction. Upon oligomerization of apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1 becomes active and cleaves the proinflammatory cytokine IL-1ß into its active secreted form. Hydrogen sulfide (H2S), a gasotransmitter mainly produced by cystathionine γ-lyase (CSE) in macrophages, could modulate inflammation. Here, we sought to investigate the effects of exogenous and endogenous H2S on NLRP3 inflammasome activation in vitro and in vivo Primed bone marrow-derived macrophages (BMDM) isolated from wildtype (wt) or CSE-deficient mice and human macrophages (THP1 cells and primary macrophages), were stimulated with MSU crystals in the presence or absence of a H2S donor, sodium thiosulfate (STS) or GYY4137 (GYY). In murine and human macrophages in vitro, both STS and GYY inhibited MSU crystal-induced IL-1ß secretion in a dose-dependent manner. Moreover, the H2S donors inhibited MSU crystal-induced XO/caspase-1 activities, mitochondrial reactive oxygen species (ROS) generation, and ASC oligomerization. Accordingly, IL-1ß secretion and XO/caspase-1 activities were higher in CSE-deficient BMDMs than in wt BMDMs. For in vivo studies, we experimentally induced peritonitis by intraperitoneal injection of MSU crystals into mice. GYY pretreatment ameliorated inflammation, evidenced by decreased IL-6/monocyte chemoattractant protein-1 (MCP-1) released into peritoneal lavages. Taken together, our results suggest that both exogenous (via H2S donors) and endogenous (via CSE) H2S production may represent approaches for managing, for example, acute gout or other inflammation conditions.

Detection of ASC Oligomerization by Western Blotting.

The apoptosis-associated speck-like protein with a caspase-recruitment domain (ASC) adaptor protein bridges inflammasome sensors and caspase-1. Upon inflammasome activation, ASC nucleates in a prion-like manner into a large and single platform responsible for the recruitment and the activation of caspase-1. Active caspase-1 will in turn promote the proteolytic maturation of the pro-inflammatory cytokine IL-1ß. ASC oligomerization is a direct evidence for inflammasome activation and its detection allows a read-out independent of caspase-1 and IL-1ß. This protocol describes how to detect the oligomerization of ASC by Western blot.

Sirtuin 2 Deficiency Increases Bacterial Phagocytosis by Macrophages and Protects from Chronic Staphylococcal Infection.

Sirtuin 2 (SIRT2) is one of the seven members of the family of NAD+-dependent histone deacetylases. Sirtuins target histones and non-histone proteins according to their subcellular localization, influencing various biological processes. SIRT2 resides mainly in the cytoplasm and regulates cytoskeleton dynamics, cell cycle, and metabolic pathways. As such, SIRT2 has been implicated in the pathogenesis of neurodegenerative, metabolic, oncologic, and chronic inflammatory disorders. This motivated the development of SIRT2-directed therapies for clinical purposes. However, the impact of SIRT2 on antimicrobial host defense is largely unknown. Here, we address this question using SIRT2 knockout mice. We show that SIRT2 is the most highly expressed sirtuin in myeloid cells, especially macrophages. SIRT2 deficiency does not affect immune cell development and marginally impacts on intracellular signaling and cytokine production by splenocytes and macrophages. However, SIRT2 deficiency enhances bacterial phagocytosis by macrophages. In line with these observations, in preclinical models, SIRT2 deficiency increases survival of mice with chronic staphylococcal infection, while having no effect on the course of toxic shock syndrome toxin-1, LPS or TNF-induced shock, fulminant Escherichia coli peritonitis, sub-lethal Klebsiella pneumoniae pneumonia, and chronic candidiasis. Altogether, these data support the safety profile of SIRT2 inhibitors under clinical development in terms of susceptibility to infections.

Sirtuin 3 deficiency does not alter host defenses against bacterial and fungal infections.

Sirtuin 3 (SIRT3) is the main mitochondrial deacetylase. SIRT3 regulates cell metabolism and redox homeostasis, and protects from aging and age-associated pathologies. SIRT3 may drive both oncogenic and tumor-suppressive effects. SIRT3 deficiency has been reported to promote chronic inflammation-related disorders, but whether SIRT3 impacts on innate immune responses and host defenses against infections remains essentially unknown. This aspect is of primary importance considering the great interest in developing SIRT3-targeted therapies. Using SIRT3 knockout mice, we show that SIRT3 deficiency does not affect immune cell development and microbial ligand-induced proliferation and cytokine production by splenocytes, macrophages and dendritic cells. Going well along with these observations, SIRT3 deficiency has no major impact on cytokine production, bacterial burden and survival of mice subjected to endotoxemia, Escherichia coli peritonitis, Klebsiella pneumoniae pneumonia, listeriosis and candidiasis of diverse severity. These data suggest that SIRT3 is not critical to fight infections and support the safety of SIRT3-directed therapies based on SIRT3 activators or inhibitors for treating metabolic, oncologic and neurodegenerative diseases without putting patients at risk of infection.

Pyrrolidine dithiocarbamate administered during ex-vivo lung perfusion promotes rehabilitation of injured donor rat lungs obtained after prolonged warm ischemia.

Damaged lung grafts obtained after circulatory death (DCD lungs) and warm ischemia may be at high risk of reperfusion injury after transplantation. Such lungs could be pharmacologically reconditioned using ex-vivo lung perfusion (EVLP). Since acute inflammation related to the activation of nuclear factor kappaB (NF-κB) is instrumental in lung reperfusion injury, we hypothesized that DCD lungs might be treated during EVLP by pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB. Rat lungs exposed to 1h warm ischemia and 2 h cold ischemia were subjected to EVLP during 4h, in absence (CTRL group, N = 6) or in presence of PDTC (2.5g/L, PDTC group, N = 6). Static pulmonary compliance (SPC), peak airway pressure (PAWP), pulmonary vascular resistance (PVR), and oxygenation capacity were determined during EVLP. After EVLP, we measured the weight gain of the heart-lung block (edema), and the concentration of LDH (cell damage), proteins (permeability edema) and of the cytokines IL-6, TNF-α and CINC-1 in bronchoalveolar lavage (BAL), and we evaluated NF-κB activation by the degree of phosphorylation and degradation of its inhibitor IκBα in lung tissue. In CTRL, we found significant NF-κB activation, lung edema, and a massive release of LDH, proteins and cytokines. SPC significantly decreased, PAWP and PVR increased, while oxygenation tended to decrease. Treatment with PDTC during EVLP inhibited NF-κB activation, did not influence LDH release, but markedly reduced lung edema and protein concentration in BAL, suppressed TNFα and IL-6 release, and abrogated the changes in SPC, PAWP and PVR, with unchanged oxygenation. In conclusion, suppression of innate immune activation during EVLP using the NF-κB inhibitor PDTC promotes significant improvement of damaged rat DCD lungs. Future studies will determine if such rehabilitated lungs are suitable for in vivo transplantation.

Periodic Fever with Aphthous Stomatitis, Pharyngitis, and Cervical Adenitis Syndrome Is Associated with a CARD8 Variant Unable To Bind the NLRP3 Inflammasome.

Periodic fever with aphthous stomatitis, pharyngitis, and cervical adenitis (PFAPA) is a relatively common autoinflammatory condition that primarily affects children. Although tendencies were reported for this syndrome, genetic variations influencing risk and disease progression are poorly understood. In this study, we performed next-generation sequencing for 82 unrelated PFAPA patients and identified a frameshift variant in the CARD8 gene (CARD8-FS). Subsequently, we compared the frequency of CARD8-FS carriers in our PFAPA cohort (13.9%) with a healthy local population group (3.2%) and found a significant association between the CARD8-FS polymorphism and risk for PFAPA syndrome (p = 0.012; odds ratio: 4.96 [95% confidence interval, 1.33-18.47]). Moreover, CARD8-FS carriers display a distinct PFAPA phenotype that is characterized by a higher prevalence of symptoms out of flares and oral aphthosis (both p = 0.02 compared with PFAPA patients without the frameshift variant). CARD8 encodes a protein component of the NLRP3 inflammasome, which plays an important role in inflammation and contributes to the pathology of various autoinflammatory diseases. We found that the CARD8-FS variant led to a truncated CARD8 protein lacking the FIIND and CARD domains. As a result, the mutant CARD8 protein lost the ability to interact with the NOD domain of NLRP3. In summary, these results identify a new CARD8 variant associated with PFAPA and further suggest that disruption of the interaction between CARD8 and NLRP3 can regulate autoinflammation in patients.

AIM2 inflammasome is activated by pharmacological disruption of nuclear envelope integrity.

Inflammasomes are critical sensors that convey cellular stress and pathogen presence to the immune system by activating inflammatory caspases and cytokines such as IL-1ß. The nature of endogenous stress signals that activate inflammasomes remains unclear. Here we show that an inhibitor of the HIV aspartyl protease, Nelfinavir, triggers inflammasome formation and elicits an IL-1R-dependent inflammation in mice. We found that Nelfinavir impaired the maturation of lamin A, a structural component of the nuclear envelope, thereby promoting the release of DNA in the cytosol. Moreover, deficiency of the cytosolic DNA-sensor AIM2 impaired Nelfinavir-mediated inflammasome activation. These findings identify a pharmacologic activator of inflammasome and demonstrate the role of AIM2 in detecting endogenous DNA release upon perturbation of nuclear envelope integrity.

Toll-like receptor 5 deficiency exacerbates cardiac injury and inflammation induced by myocardial ischaemia-reperfusion in the mouse.

Myocardial ischaemia-reperfusion (MIR) triggers a sterile inflammatory response important for myocardial healing, but which may also contribute to adverse ventricular remodelling. Such inflammation is initiated by molecular danger signals released by damaged myocardium, which induce innate immune responses by activating toll-like receptors (TLRs). Detrimental roles have been recently reported for TLR2, TLR3 and TLR4. The role of other TLRs is unknown. We therefore evaluated the role of TLR5, expressed at high level in the heart, in the development of myocardial damage and inflammation acutely triggered by MIR. TLR5(-/-) and wild-type (WT) mice were exposed to MIR (30 min ischaemia, 2 h reperfusion). We measured infarct size, markers of cardiac oxidative stress, myocardial phosphorylation state of mitogen-activated protein (MAP) kinases and AKT, expression levels of chemokines and cytokines in the heart and plasma, as well as cardiac function by echography and conductance volumetry. TLR5-deficient mice had normal cardiac morphology and function under physiological conditions. After MIR, the absence of TLR5 promoted an increase in infarct size and myocardial oxidative stress. Lack of TLR5 fostered p38 phosphorylation, reduced AKT phosphorylation and markedly increased the expression of inflammatory cytokines, whereas it precipitated acute LV (left ventricle) dysfunction. Therefore, contrary to the detrimental roles of TLR2, TLR3 and TLR4 in the infarcted heart, TLR5 is important to limit myocardial damage, inflammation and functional compromise after MIR.

Cutting edge: IL-1α is a crucial danger signal triggering acute myocardial inflammation during myocardial infarction.

Myocardial infarction (MI) induces a sterile inflammatory response that contributes to adverse cardiac remodeling. The initiating mechanisms of this response remain incompletely defined. We found that necrotic cardiomyocytes released a heat-labile proinflammatory signal activating MAPKs and NF-κB in cardiac fibroblasts, with secondary production of cytokines. This response was abolished in Myd88(-/-) fibroblasts but was unaffected in nlrp3-deficient fibroblasts. Despite MyD88 dependency, the response was TLR independent, as explored in TLR reporter cells, pointing to a contribution of the IL-1 pathway. Indeed, necrotic cardiomyocytes released IL-1α, but not IL-1ß, and the immune activation of cardiac fibroblasts was abrogated by an IL-1R antagonist and an IL-1α-blocking Ab. Moreover, immune responses triggered by necrotic Il1a(-/-) cardiomyocytes were markedly reduced. In vivo, mice exposed to MI released IL-1α in the plasma, and postischemic inflammation was attenuated in Il1a(-/-) mice. Thus, our findings identify IL-1α as a crucial early danger signal triggering post-MI inflammation.

The role of oxidative stress during inflammatory processes.

Abstract The production of various reactive oxidant species in excess of endogenous antioxidant defense mechanisms promotes the development of a state of oxidative stress, with significant biological consequences. In recent years, evidence has emerged that oxidative stress plays a crucial role in the development and perpetuation of inflammation, and thus contributes to the pathophysiology of a number of debilitating illnesses, such as cardiovascular diseases, diabetes, cancer, or neurodegenerative processes. Oxidants affect all stages of the inflammatory response, including the release by damaged tissues of molecules acting as endogenous danger signals, their sensing by innate immune receptors from the Toll-like (TLRs) and the NOD-like (NLRs) families, and the activation of signaling pathways initiating the adaptive cellular response to such signals. In this article, after summarizing the basic aspects of redox biology and inflammation, we review in detail the current knowledge on the fundamental connections between oxidative stress and inflammatory processes, with a special emphasis on the danger molecule high-mobility group box-1, the TLRs, the NLRP-3 receptor, and the inflammasome, as well as the transcription factor nuclear factor-κB.

The sirtuin inhibitor cambinol impairs MAPK signaling, inhibits inflammatory and innate immune responses and protects from septic shock.

Sirtuins (SIRT1-7) are NAD(+)-dependent histone deacetylases (HDACs) that play an important role in the control of metabolism and proliferation and the development of age-associated diseases like oncologic, cardiovascular and neurodegenerative diseases. Cambinol was originally described as a compound inhibiting the activity of SIRT1 and SIRT2, with efficient anti-tumor activity in vivo. Here, we studied the effects of cambinol on microbial sensing by mouse and human immune cells and on host innate immune responses in vivo. Cambinol inhibited the expression of cytokines (TNF, IL-1ß, IL-6, IL-12p40, and IFN-γ), NO and CD40 by macrophages, dendritic cells, splenocytes and whole blood stimulated with a broad range of microbial and inflammasome stimuli. Sirtinol, an inhibitor of SIRT1 and SIRT2 structurally related to cambinol, also decreased macrophage response to TLR stimulation. On the contrary, selective inhibitors of SIRT1 (EX-527 and CHIC-35) and SIRT2 (AGK2 and AK-7) used alone or in combination had no inhibitory effect, suggesting that cambinol and sirtinol act by targeting more than just SIRT1 and SIRT2. Cambinol and sirtinol at anti-inflammatory concentrations also did not inhibit SIRT6 activity in in vitro assay. At the molecular level, cambinol impaired stimulus-induced phosphorylation of MAPKs and upstream MEKs. Going well along with its powerful anti-inflammatory activity, cambinol reduced TNF blood levels and bacteremia and improved survival in preclinical models of endotoxic shock and septic shock. Altogether, our data suggest that pharmacological inhibitors of sirtuins structurally related to cambinol may be of clinical interest to treat inflammatory diseases.

Histone deacetylase inhibitors impair antibacterial defenses of macrophages.

Histone deacetylases (HDACs) control gene expression by deacetylating histones and nonhistone proteins. HDAC inhibitors (HDACi) are powerful anticancer drugs that exert anti-inflammatory and immunomodulatory activities. We recently reported a proof-of-concept study demonstrating that HDACi increase susceptibility to bacterial infections in vivo. Yet, still little is known about the effects of HDACi on antimicrobial innate immune defenses. Here we show that HDACi belonging to different chemical classes inhibit at multiple levels the response of macrophages to bacterial infection. HDACi reduce the phagocytosis and the killing of Escherichia coli and Staphylococcus aureus by macrophages. In line with these findings, HDACi decrease the expression of phagocytic receptors and inhibit bacteria-induced production of reactive oxygen and nitrogen species by macrophages. Consistently, HDACi impair the expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits and inducible nitric oxide synthase. These data indicate that HDACi have a strong impact on critical antimicrobial defense mechanisms in macrophages.

Histone deacetylase inhibitors impair innate immune responses to Toll-like receptor agonists and to infection.

Regulated by histone acetyltransferases and deacetylases (HDACs), histone acetylation is a key epigenetic mechanism controlling chromatin structure, DNA accessibility, and gene expression. HDAC inhibitors induce growth arrest, differentiation, and apoptosis of tumor cells and are used as anticancer agents. Here we describe the effects of HDAC inhibitors on microbial sensing by macrophages and dendritic cells in vitro and host defenses against infection in vivo. HDAC inhibitors down-regulated the expression of numerous host defense genes, including pattern recognition receptors, kinases, transcription regulators, cytokines, chemokines, growth factors, and costimulatory molecules as assessed by genome-wide microarray analyses or innate immune responses of macrophages and dendritic cells stimulated with Toll-like receptor agonists. HDAC inhibitors induced the expression of Mi-2ß and enhanced the DNA-binding activity of the Mi-2/NuRD complex that acts as a transcriptional repressor of macrophage cytokine production. In vivo, HDAC inhibitors increased the susceptibility to bacterial and fungal infections but conferred protection against toxic and septic shock. Thus, these data identify an essential role for HDAC inhibitors in the regulation of the expression of innate immune genes and host defenses against microbial pathogens.

Histone deacetylase inhibitors repress macrophage migration inhibitory factor (MIF) expression by targeting MIF gene transcription through a local chromatin deacetylation.

The cytokine macrophage migration inhibitory factor plays a central role in inflammation, cell proliferation and tumorigenesis. Moreover, macrophage migration inhibitory factor levels correlate with tumor aggressiveness and metastatic potential. Histone deacetylase inhibitors are potent antitumor agents recently introduced in the clinic. Therefore, we hypothesized that macrophage migration inhibitory factor would represent a target of histone deacetylase inhibitors. Confirming our hypothesis, we report that histone deacetylase inhibitors of various chemical classes strongly inhibited macrophage migration inhibitory factor expression in a broad range of cell lines, in primary cells and in vivo. Nuclear run on, transient transfection with macrophage migration inhibitory factor promoter reporter constructs and transduction with macrophage migration inhibitory factor expressing adenovirus demonstrated that trichostatin A (a prototypical histone deacetylase inhibitor) inhibited endogenous, but not episomal, MIF gene transcription. Interestingly, trichostatin A induced a local and specific deacetylation of macrophage migration inhibitory factor promoter-associated H3 and H4 histones which did not affect chromatin accessibility but was associated with an impaired recruitment of RNA polymerase II and Sp1 and CREB transcription factors required for basal MIF gene transcription. Altogether, this study describes a new molecular mechanism by which histone deacetylase inhibitors inhibit MIF gene expression, and suggests that macrophage migration inhibitory factor inhibition by histone deacetylase inhibitors may contribute to the antitumorigenic effects of histone deacetylase inhibitors.