G-CSF suppresses allergic pulmonary inflammation, downmodulating cytokine, chemokine and eosinophil production

AimsGranulocyte Colony-Stimulating Factor (G-CSF), which mobilizes hemopoietic stem cells (HSC), is believed to protect HSC graft recipients from graft-versus-host disease by enhancing Th2 cytokine secretion. Accordingly, G-CSF should aggravate Th2-dependent allergic pulmonary inflammation and the associated eosinophilia. We evaluated the effects of G-CSF in a model of allergic pulmonary inflammation.Main methodsAllergic pulmonary inflammation was induced by repeated aerosol allergen challenge in ovalbumin-sensitized C57BL/6J mice. The effects of allergen challenge and of G-CSF pretreatment were evaluated by monitoring: a) eosinophilia and cytokine/chemokine content of bronchoalveolar lavage fluid, pulmonary interstitium, and blood; b) changes in airway resistance; and c) changes in bone-marrow eosinophil production.Key findingsContrary to expectations, G-CSF pretreatment neither induced nor enhanced allergic pulmonary inflammation. Instead, G-CSF: a) suppressed accumulation of infiltrating eosinophils in bronchoalveolar, peribronchial and perivascular spaces of challenged lungs; and b) prevented ovalbumin challenge-induced rises in airway resistance. G-CSF had multiple regulatory effects on cytokine and chemokine production: in bronchoalveolar lavage fluid, levels of IL-1 and IL-12 (p40), eotaxin and MIP-1a were decreased; in plasma, KC, a neutrophil chemoattractant, was increased, while IL-5 was decreased and eotaxin was unaffected. In bone-marrow, G-CSF: a) prevented the increase in bone-marrow eosinophil production induced by ovalbumin challenge of sensitized mice; and b) selectively stimulated neutrophil colony formation.SignificanceThese observations challenge the view that G-CSF deviates cytokine production towards a Th2 profile in vivo, and suggest that this neutrophil-selective hemopoietin affects eosinophilic inflammation by a combination of effects on lung cytokine production and bone-marrow hemopoiesis.     

Dietary cholesterol induces trafficking of intestinal Niemann-Pick Type C1 Like 1 from the brush border to endosomes

The transmembrane protein Niemann-Pick C1 Like 1 (NPC1L1) belongs to the Niemann-Pick C1 (NPC1) family of cholesterol transporters and is mainly expressed in the liver and the small intestine. NPC1L1 is believed to be the main transporter responsible for the absorption of dietary cholesterol. Like NPC1, NPC1L1 contains a sterol sensing domain, suggesting that it might be sensitive to dietary cholesterol. To test this hypothesis, mucosal explants were cultured in the presence or absence of cholesterol. In the absence of cholesterol NPC1L1 was localized mainly in the brush border of the enterocyte, colocalizing with the brush border enzyme aminopeptidase N (APN), and only a minor part was present in intracellular compartments. In contrast, following culture in the presence of cholesterol a major part of NPC1L1 was found in intracellular compartments positive for the early endosomal marker early endosome antigen 1, whereas only a minor fraction was left in the brush border. Neither APN, lactase, nor sucrase-isomaltase was endocytosed in parallel, demonstrating that this is a selective cholesterol-induced endocytosis of NPC1L1. Conceivably either the induced internalization could be due to NPC1L1 acting as an endocytic cholesterol receptor or it could be a mechanism to reduce the cholesterol uptake. The fluorescent cholesterol analog NBD-cholesterol readily labeled the cytoplasm also under conditions nonpermissible for endocytosis, arguing against a receptor-mediated uptake. We therefore propose that cholesterol is absorbed by NPC1L1 acting as a membrane transporter and that NPC1L1 is internalized to an endosomal compartment to reduce the absorption of cholesterol.     

Discriminating multi-species populations in biofilms with peptide nucleic acid fluorescence in situ hybridization (PNA FISH)

Background

Our current understanding of biofilms indicates that these structures are typically composed of many different microbial species. However, the lack of reliable techniques for the discrimination of each population has meant that studies focusing on multi-species biofilms are scarce and typically generate qualitative rather than quantitative data.

Methodology/Principal Findings

We employ peptide nucleic acid fluorescence in situ hybridization (PNA FISH) methods to quantify and visualize mixed biofilm populations. As a case study, we present the characterization of Salmonella enterica/Listeria monocytogenes/Escherichia coli single, dual and tri-species biofilms in seven different support materials. Ex-situ, we were able to monitor quantitatively the populations of ∼56 mixed species biofilms up to 48 h, regardless of the support material. In situ, a correct quantification remained more elusive, but a qualitative understanding of biofilm structure and composition is clearly possible by confocal laser scanning microscopy (CLSM) at least up to 192 h. Combining the data obtained from PNA FISH/CLSM with data from other established techniques and from calculated microbial parameters, we were able to develop a model for this tri-species biofilm. The higher growth rate and exopolymer production ability of E. coli probably led this microorganism to outcompete the other two [average cell numbers (cells/cm²) for 48 h biofilm: E. coli 2,1×10⁸ (±2,4×10⁷); L. monocytogenes 6,8×10⁷ (±9,4×10⁶); and S. enterica 1,4×10⁶ (±4,1×10⁵)]. This overgrowth was confirmed by CSLM, with two well-defined layers being easily identified: the top one with E. coli, and the bottom one with mixed regions of L. monocytogenes and S. enterica.

Significance

While PNA FISH has been described previously for the qualitative study of biofilm populations, the present investigation demonstrates that it can also be used for the accurate quantification and spatial distribution of species in polymicrobial communities. Thus, it facilitates the understanding of interspecies interactions and how these are affected by changes in the surrounding environment.     

Effects of landscape and patch attributes on the functional diversity of medium and large-sized mammals in the Brazilian Cerrado

The Cerrado ecoregion, although threatened, holds an important diversity of medium- and large-sized mammals, which play considerable ecological functions. However, the functional diversity of medium- and large-sized mammals is considerably affected by landscape characteristics. Thus, we inventoried medium- and large-sized mammals in nine patches (natural forest remnants) in the Cerrado ecoregion of central Brazil and investigated the influence of patch and landscape attributes (buffer with 5 km around patch) on their functional diversity. We surveyed these species from August 2016 to January 2017 through camera trapping and active search techniques (tracks, vocalizations, and burrows). Functional diversity was calculated based on body mass, behavior (presence or absence of social behavior), type of locomotion, dietary habits, and environmental sensitivity. For analysis, we divided species into two groups: forest dependent mammals (FDM) and forest non-dependent mammals (NDM). We used Akaike’s information criterion corrected for small sample size to select the models that best explain the functional diversity at the local (patch) and landscape scales. The FDM species were influenced by Normalized Difference Vegetation Index (NDVI) and patch size at the patch scale and by mean patch size and edge density at the landscape scale. The NDM were not influenced by patch and landscape scale attributes. Our results indicated that analyses at two scales with the mammal species grouped considering their differences in dependence of forest remnants is a valid approach for evaluating diversity patterns of medium-and large-sized mammals in the Brazilian Cerrado ecoregion.

     

NLRP3 Controls Trypanosoma cruzi Infection through a Caspase-1-Dependent IL-1R-Independent NO Production

Trypanosoma cruzi (T. cruzi) is an intracellular protozoan parasite and the etiological agent of Chagas disease, a chronic infectious illness that affects millions of people worldwide. Although the role of TLR and Nod1 in the control of T. cruzi infection is well-established, the involvement of inflammasomes remains to be elucidated. Herein, we demonstrate for the first time that T. cruzi infection induces IL-1β production in an NLRP3- and caspase-1-dependent manner. Cathepsin B appears to be required for NLRP3 activation in response to infection with T. cruzi, as pharmacological inhibition of cathepsin B abrogates IL-1β secretion. NLRP3^−/− and caspase1^−/− mice exhibited high numbers of T. cruzi parasites, with a magnitude of peak parasitemia comparable to MyD88^−/− and iNOS^−/− mice (which are susceptible models for T. cruzi infection), indicating the involvement of NLRP3 inflammasome in the control of the acute phase of T. cruzi infection. Although the inflammatory cytokines IL-6 and IFN-γ were found in spleen cells from NLRP3^−/− and caspase1^−/− mice infected with T. cruzi, these mice exhibited severe defects in nitric oxide (NO) production and an impairment in macrophage-mediated parasite killing. Interestingly, neutralization of IL-1β and IL-18, and IL-1R genetic deficiency demonstrate that these cytokines have a minor effect on NO secretion and the capacity of macrophages to control T. cruzi infection. In contrast, inhibition of caspase-1 with z-YVAD-fmk abrogated NO production by WT and MyD88^−/− macrophages and rendered them as susceptible to T. cruzi infection as NLRP3^−/− and caspase-1^−/− macrophages. Taken together, our results demonstrate a role for the NLRP3 inflammasome in the control of T. cruzi infection and identify NLRP3-mediated, caspase-1-dependent and IL-1R-independent NO production as a novel effector mechanism for these innate receptors.     

Biology of the TAM Receptors

The TAM receptors—Tyro3, Axl, and Mer—comprise a unique family of receptor tyrosine kinases, in that as a group they play no essential role in embryonic development. Instead, they function as homeostatic regulators in adult tissues and organ systems that are subject to continuous challenge and renewal throughout life. Their regulatory roles are prominent in the mature immune, reproductive, hematopoietic, vascular, and nervous systems. The TAMs and their ligands—Gas6 and Protein S—are essential for the efficient phagocytosis of apoptotic cells and membranes in these tissues; and in the immune system, they act as pleiotropic inhibitors of the innate inflammatory response to pathogens. Deficiencies in TAM signaling are thought to contribute to chronic inflammatory and autoimmune disease in humans, and aberrantly elevated TAM signaling is strongly associated with cancer progression, metastasis, and resistance to targeted therapies.The name of the TAM family is derived from the first letter of its three constituents—Tyro3, Axl, and Mer (Prasad et al. 2006). As detailed in Figure 1, members of this receptor tyrosine kinase (RTK) family were independently identified by several different groups and appear in the early literature under multiple alternative names. However, Tyro3, Axl, and Mer (officially c-Mer or MerTK for the protein, Mertk for the gene) have now been adopted as the NCBI designations. The TAMs were first grouped into a distinct RTK family (the Tyro3/7/12 cluster) in 1991, through PCR cloning of their kinase domains (Lai and Lemke 1991). The isolation of full-length cDNAs for Axl (O''Bryan et al. 1991), Mer (Graham et al. 1994), and Tyro3 (Lai et al. 1994) confirmed their segregation into a structurally distinctive family of orphan RTKs (Manning et al. 2002b). The two ligands that bind and activate the TAMs—Gas6 and Protein S (Pros1)—were identified shortly thereafter (Ohashi et al. 1995; Stitt et al. 1995; Mark et al. 1996; Nagata et al. 1996).Open in a separate windowFigure 1.TAM receptors and ligands. The TAM receptors (red) are Tyro3 (Lai and Lemke 1991; Lai et al. 1994)—also designated Brt (Fujimoto and Yamamoto 1994), Dtk (Crosier et al. 1994), Rse (Mark et al. 1994), Sky (Ohashi et al. 1994), and Tif (Dai et al. 1994); Axl (O''Bryan et al. 1991)—also designated Ark (Rescigno et al. 1991), Tyro7 (Lai and Lemke 1991), and Ufo (Janssen et al. 1991); and Mer (Graham et al. 1994)—also designated Eyk (Jia and Hanafusa 1994), Nyk (Ling and Kung 1995), and Tyro12 (Lai and Lemke 1991). The TAMs are widely expressed by cells of the mature immune, nervous, vascular, and reproductive systems. The TAM ligands (blue) are Gas6 and Protein S (Pros1). The carboxy-terminal SHBG domains of the ligands bind to the immunoglobulin (Ig) domains of the receptors, induce dimerization, and activate the TAM tyrosine kinases. When γ-carboxylated in a vitamin-K-dependent reaction, the amino-terminal Gla domains of the dimeric ligands bind to the phospholipid phosphatidylserine expressed on the surface on an apposed apoptotic cell or enveloped virus. See text for details. (From Lemke and Burstyn-Cohen 2010; adapted, with permission, from the authors.)Subsequent progress on elucidating the biological roles of the TAM receptors was considerably slower and ultimately required the derivation of mouse loss-of-function mutants (Camenisch et al. 1999; Lu et al. 1999). The fact that Tyro3^−/−, Axl^−/−, and Mer^−/− mice are all viable and fertile permitted the generation of a complete TAM mutant series that included all possible double mutants and even triple mutants that lack all three receptors (Lu et al. 1999). Remarkably, these Tyro3^−/−Axl^−/−Mer^−/− triple knockouts (TAM TKOs) are viable, and for the first 2–3 wk after birth, superficially indistinguishable from their wild-type counterparts (Lu et al. 1999). Because many RTKs play essential roles in embryonic development, even single loss-of-function mutations in RTK genes often result in an embryonic-lethal phenotype (Gassmann et al. 1995; Lee et al. 1995; Soriano 1997; Arman et al. 1998). The postnatal viability of mice in which an entire RTK family is ablated completely—the TAM TKOs can survive for more than a year (Lu et al. 1999)—is therefore highly unusual. Their viability notwithstanding, the TAM mutants go on to develop a plethora of phenotypes, some of them debilitating (Camenisch et al. 1999; Lu et al. 1999; Lu and Lemke 2001; Scott et al. 2001; Duncan et al. 2003; Prasad et al. 2006). Almost without exception, these phenotypes are degenerative in nature and reflect the loss of TAM signaling activities in adult tissues that are subject to regular challenge, renewal, and remodeling. These activities are the subject of this review.     

Pro-inflammatory cytokines can act as intracellular modulators of commensal bacterial virulence

Interactions between commensal pathogens and hosts are critical for disease development but the underlying mechanisms for switching between the commensal and virulent states are unknown. We show that the human pathogen Neisseria meningitidis, the leading cause of pyogenic meningitis, can modulate gene expression via uptake of host pro-inflammatory cytokines leading to increased virulence. This uptake is mediated by type IV pili (Tfp) and reliant on the PilT ATPase activity. Two Tfp subunits, PilE and PilQ, are identified as the ligands for TNF-α and IL-8 in a glycan-dependent manner, and their deletion results in decreased virulence and increased survival in a mouse model. We propose a novel mechanism by which pathogens use the twitching motility mode of the Tfp machinery for sensing and importing host elicitors, aligning with the inflamed environment and switching to the virulent state.