ROS sets the stage for macrophage differentiation

While M1 macrophages are highly pro-inflammatory and microbicidal, M2 macrophages and the related tumor associated macrophages (TAMs) regulate tissue remodeling and angiogenesis and can display immunomodulatory activity. In July issue of Cell Research, Zhang et al. show that ROS production, critical for the activation and functions of M1 macrophages, is necessary for the differentiation of M2 macrophages and TAMs, and that antioxidant therapy blocks TAM differentiation and tumorigenesis in mouse models of cancer.Macrophages are key orchestrators in both the initiation and resolution stages of inflammation, and function as sentinel cells that maintain homeostasis and protect against infection. They are activated by many stimuli including pathogen-associated molecular patterns (PAMPs), endogenous danger-associated molecular patterns (DAMPs), and cytokines found in the tissue microenvironment¹. During their activation, macrophages can polarize to pro-inflammatory or anti-inflammatory states with distinct phenotypes and physiological responses — the classical pro-inflammatory M1 state induced by LPS and interferon-γ (IFN-γ) and the “alternative” M2 state triggered by IL-4 and IL-13². The M1 state is characterized by increased expression of pro-inflammatory cytokines as well as microbicidal activity, while M2 macrophages upregulate the anti-inflammatory cytokine IL-10 and participate in tissue remodeling, wound repair, and host defense against large parasites.M2-like macrophage polarization is of particular pathophysiological consequence in the setting of cancer. Early in tumor development, monocytes are recruited by tumor and stromal cell-derived chemokines to take up residence at the tumor site, where they differentiate into macrophages in response to MCSF produced by tumor cells. Such tumor-associated macrophages (TAMs) facilitate multiple steps in tumorigenesis, including promotion of tumor cell proliferation and resistance to apoptosis as well as secretion of pro-angiogenic factors and proteolytic enzymes that aid tumor cell metastasis. TAMs also display some immunosuppressive features, such as IL-10 and TGF-β production and poor antigen presentation, which conspire to prevent tumor cell killing by infiltrating T cells. Thus, the characteristics most critical for the tumor-promoting profile of TAMs bear semblance to the M2 phenotype. Although the details of such M2 polarization are not well characterized, IL-4 produced by T-cells in the tumor, as well as other tumor-derived factors, may be critical³.In July issue of Cell Research, a study by Zhang et al.⁴ provides new insights into control of macrophage differentiation and activation. In particular, the authors show that ROS production is important in M2 but not M1 macrophage differentiation. Their experimental protocol is to treat monocytes for 6 days with M-CSF or GM-CSF to induce differentiation to macrophages, followed by polarization with IL-4 (M2 state) or LPS and IFN-γ (M1 state). Interestingly, pre-treating monocytes with the antioxidant butylated hydroxyanisole (BHA) prior to differentiation inhibits M2 but not M1 polarization, as indicated by analysis of macrophage differentiation markers and M1/M2 polarization markers. The authors attribute this to the effects of BHA, i.e., block of ROS production, in inhibiting ERK activation during macrophage differentiation, consistent with previous reports implicating a role for ROS as well as MAP kinases in macrophage differentiation⁵. Furthermore, LPS and IFN-γ but not IL-4 stimulation can “rescue” ERK activation, perhaps in a manner dependent on ROS production, thus explaining why M2 but not M1 polarization is impaired by antioxidant treatment (Figure 1).Open in a separate windowFigure 1M1 macrophages are highly pro-inflammatory and microbicidal and are polarized by treatment with LPS+IFNγ, while M2 macrophages mediate tissue repair, angiogenesis and immunomodulation. Tumor associated macrophages (TAMs), which are M2-like, are associated with worsened clinical prognosis in many cancers and are thought to be skewed by a combination of tumor-derived factors and other cytokines present in the tumor microenvironment. ROS production increases during M-CSF- or GM-CSF-induced macrophage differentiation from monocytes, and the antioxidant BHA specifically inhibits M2 and TAM polarization. LPS+IFNγ treatment is able to overcome the effects of BHA to induce normal M1 polarization, revealing a specific role for ROS in macrophage polarization.As the M2-like properties of TAMs are thought to promote tumorigenesis, Zhang et al. go on to investigate the consequences of BHA administration in mouse models of cancer. They demonstrate that in vivo treatment of BHA can attenuate cancer initiation, progression, and metastasis in multiple models. As ROS can promote tumor cell proliferation, survival, and DNA damage, BHA could be acting directly on the tumor cells to prevent growth and metastasis⁶. However, BHA had no effects on the proliferation of three tumor cell lines in vitro. The authors propose that TAM differentiation may be a critical target, as BHA administration reduced TAM numbers as well as levels of TAM markers. Moreover, in at least one of the models, BHA administration was ineffective when macrophages were depleted by clodronate injection.Collectively, the findings of Zhang et al. are intriguing for several reasons. First, ROS production is usually associated with the activation and functions of M1 rather than M2 macrophages. ROS production downstream of LPS signaling mediates production of pro-inflammatory cytokines (in part through MAP kinase activation). ROS and nitric oxide (NO) production by NADPH oxidase and iNOS, respectively, as well as mROS upregulation are key to the antimicrobial activity of M1 macrophages⁷. Indeed NO production can inhibit oxidative metabolism, pivotal to the survival and function of M2 macrophages⁸. Thus ROS production may be important in M1 activation and function while the requirement for ROS in M2 differentiation may be most critical during MCSF-mediated differentiation rather than IL-4-triggered polarization. Future studies to better understand the role of ROS production in macrophage differentiation and activation may be informative. Second, it would be interesting to further probe the effects of BHA in inhibiting tumorigenesis. The authors'' in vitro studies suggest inhibition of TAM differentiation as one underlying mechanism, but one can envision additional possibilities. At least in some cancers, tumor cells and other immune cells in the microenvironment produce ROS that promote inflammation⁹, thus contributing to tumorigenesis. mROS has been linked to activation of HIF1α, which can facilitate angiogenesis and metastasis. Indeed, it is worth pointing out that ROS can regulate many cellular processes, some of which have already been alluded to, including signal transduction (e.g., downstream of growth factor receptors and innate immune signaling pathways as well as MAP kinase activation), redox signaling, autophagy, and respiratory burst and other antimicrobial activities¹⁰. Thus it is likely that other cellular processes perturbed by antioxidant treatment contribute to the effects of BHA in reducing tumorigenesis.Finally, the study by Zhang et al. suggests that treatment with BHA or perhaps other antioxidants could be considered in therapeutic control of cancer. Indeed, there is tremendous interest in the clinical use of antioxidants for treating many diseases. Given the pleiotropic activities of ROS mentioned above, it would be important to better understand the molecular pathways by which antioxidants exert their effects.     

Effects of age,territoriality and breeding on survival of Bonelli’s Eagle Aquila fasciata

Survival typically contributes most to population trends in long‐lived birds and its accurate estimation is therefore vital for population management and conservation. We evaluated the effects of age, territoriality and reproduction on survival in Bonelli’s Eagle Aquila fasciata through multistate capture‐mark‐recapture analyses on a long‐term dataset. Monitoring was carried out in southeast France (1990–2008) and involved the surveying of territorial Eagles, the marking of fledged chicks, and the recording of resightings and recoveries of marked non‐territorial and territorial birds. Survival improved with age, but territoriality was not retained in the best model; yearly survival was estimated at 0.479 for fledglings (to 1 year of age), 0.570 for 1‐ and 2‐year‐olds, and 0.870 for 3‐year‐old and older individuals. The second best model supported a further increase in survival from 3‐year‐olds (0.821) to older individuals (0.880). In the third best supported model, territoriality enhanced survival, but only in 2‐year‐olds (0.632 vs. 0.562 for non‐territorial). We found no correlation between the previous breeding stage and future survival, consistent with the long lifespan of the study species. Nevertheless, 4‐year‐old and older successful breeders were more likely to breed the following year than failed adult breeders (0.869 vs. 0.582), suggesting that the cost of reproduction is small in comparison with the variation in quality among individuals or their territories.     

A new Late Jurassic turtle from Spain: phylogenetic implications,taphonomy and palaeoecology

Abstract: The Jurassic was an important period in the evolution of Testudinata and encompasses the origin of many clades, and this is especially true of Jurassic turtles from Western Europe. A new genus and species of Late Jurassic turtle, Hispaniachelys prebetica gen. et sp. nov. from the upper Oxfordian of the Prebetic (Southern Spain), is described on the basis of postcranial material. The specimen is the only known tetrapod from the Mesozoic of the Prebetic and the oldest turtle from southern Europe. A mosaic of characters indicates this is a new genus: it displays basal features including dorsal epiplastral processes/reduced cleithra, no medial contact of the extragulars and a long first thoracic rib, alongside derived characters including an absence of mesoplastra and the vertebral 3/4 sulcus crossing neural 5. The phylogenetic position of the new taxon is hard to resolve, and it might be either a paracryptodire or a basal testudine, but it is distinct from Plesiochelys. A complex taphonomic history is shown by a range of overlying grazing traces and bioerosion on the carapace. The carapace was subsequently overturned and buried ventrally up, terminating grazing activity, and was then bored by sponges before final burial. Scanning electron microscopy reveals phosphatic microspheroids associated with bacterial decay in the vascular cavities of the cancellous bone, suggesting the carapace may have acted as a closed microenvironment in which decay‐derived authigenic minerals formed.     

Proline-rich cell wall proteins accumulate in growing regions and phloem tissue in response to water deficit in common bean seedlings

Plant cell walls undergo dynamic changes in response to different environmental stress conditions. In response to water deficit, two related proline-rich glycoproteins, called p33 and p36, accumulate in the soluble fraction of the cell walls in Phaseolus vulgaris (Covarrubias et al. in Plant Physiol 107:1119–1128, 1995). In this work, we show that p33 and p36 are able to form a 240 kDa oligomer, which is found in the cell wall soluble fraction. We present evidence indicating that the highest accumulation of these proteins in response to water deficit occurs in the growing regions of common bean seedlings, particularly in the phloem tissues. These proteins were detected in P. vulgaris cell suspension cultures, where the p33/p36 ratio was higher under hyperosmotic conditions than in bean seedlings subjected to the same treatment. The results support a role for these proteins during the plant cell response to changes in its water status, and suggest that cell wall modifications are induced in active growing cells of common bean in response to water limitation. Marina Battaglia and Rosa M. Solórzano contributed equally to this work.     

Interaction with host factors exacerbates Trypanosoma cruzi cell invasion capacity upon oral infection

Outbreaks of severe acute Chagas’ disease acquired by oral infection, leading to death in some cases, have occurred in recent years. Using the mouse model, we investigated the basis of such virulence by analyzing a Trypanosoma cruzi isolate, SC, from a patient with severe acute clinical symptoms, who was infected by oral route. It has previously been shown that, upon oral inoculation into mice, T. cruzi metacyclic trypomastigotes invade the gastric mucosal epithelium by engaging the stage-specific surface glycoprotein gp82, whereas the surface molecule gp90 functions as a down-modulator of cell invasion. We found that, when orally inoculated into mice, metacyclic forms of the SC isolate, which express high levels of gp90, produced high parasitemias and high mortality, in sharp contrast with the reduced infectivity in vitro. Upon recovery from the mouse stomach 1 h after oral inoculation, the gp90 molecule of the parasites was completely degraded, and their entry into HeLa cells, as well as into Caco-2 cells, was increased. The gp82 molecule was more resistant to digestive action of the gastric juice. Host cell invasion of SC isolate metacyclic trypomastigotes was augmented in the presence of gastric mucin. No alteration in infectivity was observed in T. cruzi strains CL and G which were used as references and which express gp90 molecules resistant to degradation by gastric juice. Taken together, our findings suggest that the exacerbation of T. cruzi infectivity, such as observed upon interaction of the SC isolate with the mouse stomach components, may be responsible for the severity of acute Chagas’ disease that has been reported in outbreaks of oral T. cruzi infection.     

Zn2+-dependent redox switch in the intracellular T1-T1 interface of a Kv channel

The thiol-based redox regulation of proteins plays a central role in cellular signaling. Here, we investigated the redox regulation at the Zn(2+) binding site (HX(5)CX(20)CC) in the intracellular T1-T1 inter-subunit interface of a Kv4 channel. This site undergoes conformational changes coupled to voltage-dependent gating, which may be sensitive to oxidative stress. The main results show that internally applied nitric oxide (NO) inhibits channel activity profoundly. This inhibition is reversed by reduced glutathione and suppressed by intracellular Zn(2+), and at least two Zn(2+) site cysteines are required to observe the NO-induced inhibition (Cys-110 from one subunit and Cys-132 from the neighboring subunit). Biochemical evidence suggests strongly that NO induces a disulfide bridge between Cys-110 and Cys-132 in intact cells. Finally, further mutational studies suggest that intra-subunit Zn(2+) coordination involving His-104, Cys-131, and Cys-132 protects against the formation of the inhibitory disulfide bond. We propose that the interfacial T1 Zn(2+) site of Kv4 channels acts as a Zn(2+)-dependent redox switch that may regulate the activity of neuronal and cardiac A-type K(+) currents under physiological and pathological conditions.     

Mechanistic Insights into the Modulation of Voltage-Gated Ion Channels by Inhalational Anesthetics

General anesthesia is a relatively safe medical procedure, which for nearly 170 years has allowed life saving surgical interventions in animals and people. However, the molecular mechanism of general anesthesia continues to be a matter of importance and debate. A favored hypothesis proposes that general anesthesia results from direct multisite interactions with multiple and diverse ion channels in the brain. Neurotransmitter-gated ion channels and two-pore K⁺ channels are key players in the mechanism of anesthesia; however, new studies have also implicated voltage-gated ion channels. Recent biophysical and structural studies of Na⁺ and K⁺ channels strongly suggest that halogenated inhalational general anesthetics interact with gates and pore regions of these ion channels to modulate function. Here, we review these studies and provide a perspective to stimulate further advances.     

Interplay between HIV-1 infection and host microRNAs

Using microRNA array analyses of in vitro HIV-1-infected CD4(+) cells, we find that several host microRNAs are significantly up- or downregulated around the time HIV-1 infection peaks in vitro. While microRNA-223 levels were significantly enriched in HIV-1-infected CD4(+)CD8(-) PBMCs, microRNA-29a/b, microRNA-155 and microRNA-21 levels were significantly reduced. Based on the potential for microRNA binding sites in a conserved sequence of the Nef-3'-LTR, several host microRNAs potentially could affect HIV-1 gene expression. Among those microRNAs, the microRNA-29 family has seed complementarity in the HIV-1 3'-UTR, but the potential suppressive effect of microRNA-29 on HIV-1 is severely blocked by the secondary structure of the target region. Our data support a possible regulatory circuit at the peak of HIV-1 replication which involves downregulation of microRNA-29, expression of Nef, the apoptosis of host CD4 cells and upregulation of microRNA-223.     

More than multiple introductions: Multiple taxa contribute to the genesis of the invasive California’s wild artichoke thistle

The history of some invasive species is so complex that their origins can be difficult to determine. One example of such invasive species is the California invasive known as “wild artichoke thistle” (Cynara cardunculus var. sylvestris), found in natural and disturbed ecosystems. Wild artichoke thistle is a Mediterranean native and the progenitor of two domesticated horticultural taxa, artichoke and cardoon. Different hypotheses regarding the origins of California plants have included introductions by 19th century Italian immigrants and the de-domestication (evolutionary reversion to wild-type morphology) of feral (escaped, free-living) cultivars. Using microsatellite markers, we compared the genetic constitutions of 12 artichoke thistle populations in California with possible progenitor populations: 17 Spanish and Italian wild populations and eight different artichoke and cardoon cultivars. Each California population was compared with its putative progenitors using STRUCTURE analysis. Our results suggest that California's artichoke thistle populations are polyphyletic. Surprisingly, two-thirds of California's populations closely matched populations from the Iberian Peninsula. Three populations matched domesticated artichoke. One population appears to have wild and cultivar hybrid ancestry. Alleles specific to Italian populations were found at low frequencies in some California plants, suggesting that Italian wild plants may have been in California, but have left a trivial genetic legacy. Given that the de-domesticated plants in this study appear to be as invasive as the wild taxon, we conclude with a discussion of the role that ferality and de-domestication may have in plant invasions.     

Effects of reinforcer quality and step size on rats' performance under progressive ratio schedules

The Mathematical Principles of Reinforcement (MPR) model accurately predicts performance in fixed ratio (FR) schedules of reinforcement. The aim of the present study was to assess the generality of MPR with rats responding under progressive ratio (PR) schedules of different step sizes (PR1 or PR3) that provided either food or saccharin pellets. The results showed that the rats responding for saccharin pellets produced higher breakpoints (i.e., completed higher ratios) than those responding for food pellets. In terms of theoretical parameters, one finding unanticipated on the basis of MPR was that the a parameter (specific activation) was higher for the PR3 schedules. This finding suggests that specific activation may be affected indirectly by motor parameters of the task.