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Macrophages are active contributors to the innate immune defense system. As macrophage activation is clearly affected by the surrounding microenvironment, the present study investigated the effect of sulforaphane (SFN) on the bactericidal activity of macrophages and the underlying molecular mechanisms involved in this process. Human THP‑1‑derived macrophages, primary human peripheral blood mononuclear cell‑derived macrophages, and primary mouse bone marrow derived‑macrophages (BMDMs) pretreated with SFN or DMSO were utilized in a model of Staphylococcus aureus infection. The results suggested that SFN pretreatment of macrophages effectively repressed the intracellular survival of S. aureus through modulation of p38/JNK signaling and decreased S. aureus‑induced caspases‑3/7‑dependent cell apoptosis, potentially through downregulation of microRNA (miR)‑142‑5p and miR‑146a‑5p. As SFN is a well‑known activator of nuclear factor erythroid 2‑related factor 2 (Nrf2), Nrf2‑/‑ BMDMs were used to demonstrate that the SFN‑mediated inhibitory effect was independent of Nrf2. Nevertheless, an increase in intracellular bacterial survival in Nrf2‑deficient macrophages was observed. In addition, SFN pretreatment suppressed S. aureus‑induced transcriptional expression of genes coding for the proinflammatory cytokines interleukin (IL)‑1β, IL‑6, and tumor necrosis factor‑α (TNF‑α), as well as for the M1 markers C‑C motif chemokine receptor 7, IL‑23 and inducible nitric oxide synthase (iNOS). Western blot analysis indicated that S. aureus challenge activated p38 mitogen‑activated protein kinase (MAPK) (p38) and c‑Jun N‑terminal kinase (JNK) MAPK signaling pathways, while SFN pretreatment prevented p38 and JNK phosphorylation. Pretreatment with 2 specific inhibitors of p38 and JNK, SB203580 and SP600125, respectively, resulted in a decrease in S. aureus‑induced proinflammatory gene expression levels compared with those observed in the SFN‑pretreated macrophages. Furthermore, THP‑1‑derived macrophages pretreated with SB203580 or SP600125 prior to bacterial infection exhibited a significant inhibition in intracellular S. aureus survival. In conclusion, we hypothesize that concomitant targeting of the p38/JNK‑inflammatory response and the S. aureus‑induced apoptosis with SFN may be a promising therapeutic approach in S. aureus infection.

With 4 billion people in lockdown in the world, COVID-19 outbreak may result in excessive sedentary time, especially in the population of vulnerable and disabled subjects. In many chronic disorders and diseases including type 2 diabetes mellitus and hypertension, cardiovascular and immune beneficial effects of exercise interventions should be reminded.

Cervical spinal cord injury (SCI) results in permanent life-altering motor and respiratory deficits. Other than mechanical ventilation for respiratory insufficiency secondary to cervical SCI, effective treatments are lacking and the development of animal models to explore new therapeutic strategies are needed. The aim of this work was to demonstrate the feasibility of using a mouse model of partial cervical spinal hemisection at the second cervical metameric segment (C2) to investigate the impact of 6 weeks training on forced exercise wheel system on locomotor/respiratory plasticity muscles. To measure run capacity locomotor and respiratory functions, incremental exercise tests and diaphragmatic electromyography were done. In addition, muscle fiber type composition and capillary distribution were assessed at 51 days following chronic C2 injury in diaphragm, extensor digitorum communis (EDC), tibialis anterior (TA) and soleus (SOL) muscles. Six-week exercise training increased the running capacity of trained SCI mice. Fiber type composition in EDC, TA and SOL muscles was not modified by our protocol of exercise. The vascularization was increased in all muscle limbs in SCI trained group. No increase in diaphragmatic electromyography amplitude of the diaphragm muscle on the side of SCI was observed, while the contraction duration was significantly decreased in sedentary group compared to trained group. Cross-sectional area of type IIa myofiber in the contralateral diaphragm side of SCI was smaller in trained group. Fiber type distribution between contralateral and ipsilateral diaphragm in SCI sedentary group was affected, while no difference was observed in trained group. In addition, the vascularization of the diaphragm side contralateral to SCI was increased in trained group. All these results suggest an increase in fatigue resistance and a contribution to the running capacity in SCI trained group. Our exercise protocol could be a promising non-invasive strategy to sustain locomotor and respiratory muscle plasticity following SCI.

International audience; Inflammation plays a crucial role in the defense response of the innate immune system against pathogen infection. In this study, we selected 4 compounds for their potential or proven anti-inflammatory and/or anti-microbial properties to test on our in vitro model of bacteria-infected THP-1-derived macrophages. We first compared the capacity of sulforaphane (SFN), wogonin (WG), oltipraz (OTZ), and dimethyl fumarate (DMF) to induce the nuclear factor erythroid 2-related factor 2 (Nrf2), a key regulator of the antioxidant, anti-inflammatory response pathways. Next, we performed a comparative evaluation of the antioxidant and anti-inflammatory efficacies of the 4 selected compounds. THP-1-derived macrophages and LPS-stimulated macrophages were treated with each compound and expression levels of genes coding for inflammatory cytokines IL-1β, IL-6, and TNF-α were quantified by RT-qPCR. Moreover, expression levels of genes coding for M1 (IL-23, CCR7, IL-1β, IL-6, and TNF-α) and M2 (PPARγ, MRC1, CCL22, and IL-10) markers were determined in classically-activated M1 macrophages treated with each compound. Finally, the effects of each compound on the intracellular bacterial survival of gram-negative E. coli and gram-positive S. aureus in THP-1-derived macrophages and PBMC-derived macrophages were examined. Our data confirmed the anti-inflammatory and antioxidant effects of SFN, WG, and DMF on LPS-stimulated THP-1-derived macrophages. In addition, SFN or WG treatment of classically-activated THP-1-derived macrophages reduced expression levels of M1 marker genes, while SFN or DMF treatment upregulated the M2 marker gene MRC1. This decrease in expression of M1 marker genes may be correlated with the decrease in intracellular S. aureus load in SFN- or DMF-treated macrophages. Interestingly, an increase in intracellular survival of E. coli in SFN-treated THP-1-derived macrophages that was not observed in PBMC-derived macrophages. Conversely, OTZ exhibited pro-oxidant and proinflammatory properties, and affected intracellular survival of E. coli in THP-1-derived macrophages. Altogether, we provide new potential therapeutic alternatives in treating inflammation and bacterial infection.

The innate immune system, including phagocytic cells, is the first line of defense against pathogens. During infection by microorganisms such as viruses, bacteria, or parasites, phagocytic cells produce an excess of oxidants, a crucial process for the clearance of pathogens. This increase in oxidants creates an imbalance between oxidants and endogenous antioxidants. Left unchecked, this acute or chronic oxidative stress can lead to apoptotic cell-death and oxidative stress-induced diseases including neurodegenerative and cardiovascular disorders, premature aging, secondary infections, and cancer. The activation of nuclear factor E2-related factor 2 (Nrf2) is an efficient antioxidant defensive mechanism used by host cells to counteract oxidative stress. The transcription factor Nrf2 has been identified as the master regulator of several hundred of genes involved in the antioxidant defense response. The review objectives were to collect recent findings on the contribution of oxidative stress to complications of infection, and to highlight the beneficial impact of antioxidants in reducing inflammation and oxidant-related tissue damage. Furthermore, a direct relationship between infection and decline in Nrf2 activity has been demonstrated. Thus, an interesting therapeutic approach in disease prevention and treatment of stress-related diseases may consist in optimizing antibiotic or antiviral therapy with a combination of Nrf2 inducer treatment.

Mycobacterium abscessusis a pathogenic, rapidly growing mycobacterium responsible for pulmonary and cutaneous infections in immunocompetent patients and in patients with Mendelian disorders, such as cystic fibrosis (CF).Mycobacterium abscessusis known to transition from a smooth (S) morphotype with cell surface-associated glycopeptidolipids (GPL) to a rough (R) morphotype lacking GPL. Herein, we show thatM. abscessusS and R variants are able to grow inside macrophages and are present in morphologically distinct phagosomes. The S forms are found mostly as single bacteria within phagosomes characterized by a tightly apposed phagosomal membrane and the presence of an electron translucent zone (ETZ) surrounding the bacilli. By contrast, infection with the R form leads to phagosomes often containing more than two bacilli, surrounded by a loose phagosomal membrane and lacking the ETZ. In contrast to the R variant, the S variant is capable of restricting intraphagosomal acidification and induces less apoptosis and autophagy. Importantly, the membrane of phagosomes enclosing the S forms showed signs of alteration, such as breaks or partial degradation. Although not frequently encountered, these events suggest that the S form is capable of provoking phagosome–cytosol communication. In conclusion,M. abscessusS exhibits traits inside macrophages that are reminiscent of slow-growing mycobacterial species.

Pancreatic ductal adenocarcinoma is the most common pancreatic neoplasm. There are approximately 33,000 new cases of pancreatic ductal adenocarcinoma annually in the United States with approximately the same number of deaths. Surgery represents the only opportunity for cure, but this is restricted to early stage pancreatic cancer. Pancreatic ductal adenocarcinoma evolves from a progressive cascade of cellular, morphological and architectural changes from normal ductal epithelium through preneoplastic lesions termed pancreatic intraepithelial neoplasia (PanIN). These PanIN lesions are in turn associated with somatic alterations in canonical oncogenes and tumor suppressor genes. Most notably, early PanIN lesions and almost all pancreatic ductal adenocarcinomas involve mutations in the K-ras oncogene. Thus, it is believed that activating K-ras mutations are critical for initiation of pancreatic ductal carcinogenesis. This has been proven through elegant genetically engineered mouse models in which a Cre-activated K-RasG12D allele is knocked into the endogenous K-Ras locus and crossed with mice expressing Cre recombinase in pancreatic tissue. As a result, mechanistic insights are now possible into how K-Ras contributes to pancreatic ductal carcinogenesis, what cooperating events are required, and armed with this knowledge, new therapeutic approaches can be pursued and tested.

Predicting the toxic potential of compounds to the ocular surface has depended on the Draize test for the past half century. Alternatives to Draize testing have recently been sought for a number of reasons. Stress gene expression has emerged as a means of quantifying cellular reaction and, thus, the toxic potential of the compound in question. This study examines the expression of the major stress response gene heme oxygenase-1 (HO-1) in a human corneal epithelial cell line (HCE-T) following challenges with a number of known ocular irritants.HCE-T was used to investigate the effect of ocular irritants on cell viability and HO-1 expression. Irritants tested included hydrogen peroxide, isopropyl alcohol, sodium hydroxide and trichloroacetic acid. HCE-T cells were grown to 80% confluency and treated with the listed irritants at a concentration range of 10-100 microM. Cell viability and northern blot analysis were performed following a 24 and 48 hr incubation period.HCE-T cells expressed HO-1 mRNA and HO activity similar to other human cell lines. Northern blot analysis demonstrated that levels of HO-1 mRNA transcripts increased regularly after exposure to the irritants in a concentration-dependent manner. Studies on the effect of various inhibitors and inducers of HO-1 on cell viability showed that inhibition of HO-1 potentiates the cytotoxic effect of ocular irritants. In contrast, pre-induction of HO-1 in HCE-T decreases the effect of various irritants on cell viability.These results are consistent with the idea that HO-1 mRNA levels may be used as an indicator of toxicity resulting from ocular irritants and that HCE-T cells respond to stress in a fashion similar to other human cell lines. This strategy for testing may be important in the development of an alternative to Draize testing. The results of this study also suggest that HO-1 may constitute a part of the protective defense mechanism against chemical injury.

Pancreatic ductal adenocarcinoma (PDA) is an extremely aggressive malignancy, which carries a dismal prognosis. Activating mutations of the Kras gene are common to the vast majority of human PDA. In addition, recent studies have demonstrated that embryonic signaling pathway such as Hedgehog and Notch are inappropriately upregulated in this disease. The role of another embryonic signaling pathway, namely the canonical Wnt cascade, is still controversial. Here, we use gene array analysis as a platform to demonstrate general activation of the canonical arm of the Wnt pathway in human PDA. Furthermore, we provide evidence for Wnt activation in mouse models of pancreatic cancer. Our results also indicate that Wnt signaling might be activated downstream of Hedgehog signaling, which is an early event in PDA evolution. Wnt inhibition blocked proliferation and induced apoptosis of cultured adenocarcinoma cells, thereby providing evidence to support the development of novel therapeutical strategies for Wnt inhibition in pancreatic adenocarcinoma.

In a recent issue of Cell Death and Discovery, we described a new mechanism by which sulforaphane decreased mycobacterial burden in an in vitro model of THP-1-derived macrophage infection by Mycobacterium abscessus.1 This sulforaphane-mediated diminution was due to the induction of a caspase-independent cell apoptosis and necessitated activation of both nuclear factor E2-related factor 2 (Nrf2) and p38 mitogen-activated protein kinases (MAPK) signaling pathways. M. abscessus is a nontuberculous mycobacterium that belongs to the group of rapid growing mycobacteria. This emerging pathogen is able to cause skin, bone and soft tissue infections, and more generally exacerbations of lung diseases.2 Its high tolerance to antibiotics, which limits patient treatment, has become a public health concern worldwide. M. abscessus has been increasingly involved in patients with cystic fibrosis and in immunosuppressed patients,3 enhancing their risk of developing chronic airway infections and leading to a fatal outcome. Bacterial infection induces an imbalance between oxidants and antioxidants, triggering an oxidative burst that is well known for its bactericidal effect. Yet, some selected mycobacteria are able to thrive in this oxidative environment. It is then unsurprising that the use of oxidant scavengers such as MnTE-2-PyP or N-acetyl-L-cysteine has been shown to decrease M. abscessus load by activating bacterial killing in phagolysosomes.4 In our study, we decreased the oxidative environment in THP-1-derived macrophages by activating Nrf2, the key transcription factor that controls the cascade of cytoprotective and antioxidant defense mechanisms, and the maintenance of the redox homeostasis. Upon oxidative stress or infection, cytoplasmic Nrf2 is released from its inhibitor Keap-1, translocates into the nucleus, and forms a cofactor complex that binds to specific antioxidant responsive elements (ARE) found in promoters of phase II antioxidant and detoxifying enzymes. Nrf2 signaling pathway has been shown to have an important role whether beneficial or detrimental, in microbial infections.5 M. abscessus infection of THP-1-derived macrophages activates Nrf2 signaling pathway, and induces the expression of heme oxygenase-1 and NADPH quinone oxidoreductase-1, two downstream targets of Nrf2. In addition, M. abscessus induced reactive oxygen species (ROS) production in infected macrophages and blocked phagosomal acidification thus allowing its intracellular proliferation. Sulforaphane, a well-established activator of Nrf2 was used to pretreat macrophages before mycobacterial infection. The results showed an inhibition of mycobacterial growth 7 days postinfection in sulforaphane pretreated macrophages compared with vehicle pretreated ones. This sulforaphane-induced mycobacterial growth inhibition was neither caused by phagosomal pH acidification nor increase in cell phagocytosis, but rather induction of cell apoptosis. Contrary to the well-known cyprotective effect of sulforaphane and Nrf2 signaling pathway,6 we demonstrated that pre-stimulation of Nrf2 signaling pathway by sulforaphane before M. abscessus infection triggered a caspase-independent cell apoptosis. Pretreatment of macrophages with sulforaphane alone had no significant effect on cell death. To our knowledge, this is the first study showing sulforaphane-induced Nrf2 triggers a caspase3/7-independent and p38 MAPK-dependent cell apoptosis in mycobacteria infected macrophages (Figure 1). Figure 1 Infection of THP-1 derived macrophages by M. abscessus elicits caspase3/7-dependent cell apoptosis. Pretreatment of macrophages with an Nrf2 activator before mycobacterial challenge initiates a caspase-independent cell apoptosis Cell death in macrophages has a critical role in the host response to mycobacterial infection. Cell apoptosis is an effective mechanism used by the innate defense system to fight bacterial infection. This energy-dependent mechanism generates apoptotic bodies that will facilitate T-dependent antigen response and induce mycobacterial killing by uninfected neighboring macrophages.7 As M. abscessus has only been recently identified as a separate species, very little is known in the dynamics between M. abscessus and macrophage cell survival/cell death. In our in vitro model, M. abscessus induced cell necrosis in untreated cells but not cell apoptosis. This phenomenon has been observed in M. tuberculosis that has the ability to inhibit cell apoptosis in favor of cell necrosis.8 Recent highlights on caspase-independent forms of apoptosis triggered by infection have been described in the literature. Necroptosis, also known as programmed necrosis, can be initiated by various agents including microbial infection, and is characterized by activation of the receptor-interacting protein (RIP) kinases RIP1 and RIP3-dependent signaling pathway. This programmed cell death is a critical antiviral process in the host innate immune defense system. Phagocytes infected with viruses that are able to inhibit the classical caspase-dependent apoptosis pathway can undergo necroptosis to facilitate viral clearance by the neighboring immune system.9, 10 Moreover, Roca and Ramakrishnan have demonstrated a tumor-necrosis factor (TNF)-induced necroptosis in a M. tuberculosis infected zebrafish model. In the early stages of mycobacteria infection, injection of an excess of TNF in infected macrophages induced mitochondrial ROS production through a RIP1/RIP3-dependent kinase pathway, process necessary for the bacterial clearance. However, ROS-mediated necroptosis rapidly led to release and propagation of M. tuberculosis in the extracellular microenvironment.11 Another caspase 3/7-independent apoptosis is the inflammation-mediated cell death known as pyroptosis. This particular programmed cell death participates in the host cell defense system against pathogens and requires activation of caspase 1, IL-1β and IL-18.12 Recently, Bai et al.13 have shown that M. tuberculosis-mediated production of IL-32γ in THP-1-derived macrophages induced caspase 1-dependent pyroptosis which help reduce intracellular M. tuberculosis burden. In conclusion, additional studies are required to specifically identify the caspase-independent form(s) of apoptosis initiated by the combination of Nrf2 activation and M. abscessus infection. Nevertheless, our findings strongly suggest that Nrf2 activators may be of great interest as future therapeutic treatments in supplement to the actual lengthy multi-drug therapies used in patients diagnosed with mycobacterial infection.

A frequent genetic alteration found in premalignant stages of pancreatic adenocarcinoma is K-ras oncogene point mutation. The mechanistic basis for the inability of K-ras mutation to transform pancreatic ductal cells is unclear, although cooperating events with p16 inactivation, p53 mutation, and SMAD 4 mutation are recognized to be necessary. We have generated a novel mouse model in which the cytokeratin 19 promoter, specifically active in pancreatic ductal cells but not other cell types of the pancreas, is fused to mutant K-ras. This is of direct relevance to human pancreatic cancer because premalignant lesions are found specifically in ductal cells. There is dramatic periductal lymphocytic infiltration in the pancreata of transgenic mice, predominantly CD4+ T lymphocytes, which may act as an adaptive immune response to activated ras-mediated signaling. In addition, gene array analysis reveals an induction of N-cadherin in transgenic mice pancreatic ductal cells, the significance of which relates to promotion of cell adhesion and deterrence of cell migration. Apart from these important biological considerations, there is parallel activity of the cytokeratin 19 promoter in the stem cell region of the gastric epithelium, namely in mucous neck cells. Activated K-ras in this context causes mucous neck cell hyperplasia, a precursor to gastric adenocarcinoma. There is concomitant parietal cell decrease, which is a key step toward gastric adenocarcinoma. Taken together, we have defined how mutant K-ras signaling modulates important molecular events in the initiating events of pancreatic and gastric carcinogenesis.

Heme oxygenase-1 (HO-1) is a stress protein, and its induction has been suggested to participate in defense mechanisms against agents that promote oxidative injury such as endotoxins and heme. We have shown that the inflammatory cytokines, interleukin-6 (IL-6) and heme-induced HO-1 gene expression, were suppressed by dexamethasone (Dex) in a sustained manner. We examined the mechanism by which the anti-inflammatory agent, Dex, inhibits IL-6 and heme-induced HO-1 expression in rabbit coronary endothelial cells. Endothelial cells treated with heme (10 microM) and IL-6 (25 ng/ml), increased HO-1 mRNA 15- and 60-fold, respectively. The activity of HO was increased 3-fold after such treatment. Although Dex failed to inhibit heme-mediated HO-1 mRNA and HO activity, it was able to reverse IL-6-stimulated HO activity. Several human HO-1 promoter-drive chloramphenicol acetyltransferase (CAT) constructs were examined to analyze IL-6 and Dex-mediated modulation of the HO-1 gene in endothelial cells. CAT assays revealed that the HO-1 promoter region between -180 and -1500 might contain a Dex-mediated negative regulator. Gel mobility shift assays using nuclear extracts from IL-6-treated endothelial cells showed a binding to the synthetic 21 base pairs of the HO-1 sequence that contains the putative STAT3 sequence. STAT3-specific probe inhibited nuclear binding protein to the putative HO-1-STAT3 sequence. This suggests that IL-6 induction of human HO-1 is mediated via the JAK-STAT pathway and that Dex inhibition of gene expression is carried out by activation of a transcriptional protein in competition with the STAT3 binding site.