Sec22b Regulates Inflammatory Responses by Controlling the Nuclear Translocation of NF-κB and the Secretion of Inflammatory Mediators.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) regulate the vesicle transport machinery in phagocytic cells. Within the secretory pathway, Sec22b is an endoplasmic reticulum-Golgi intermediate compartment (ERGIC)-resident SNARE that controls phagosome maturation and function in macrophages and dendritic cells. The secretory pathway controls the release of cytokines and may also impact the secretion of NO, which is synthesized by the Golgi-active inducible NO synthase (iNOS). Whether ERGIC SNARE Sec22b controls NO and cytokine secretion is unknown. Using murine bone marrow-derived dendritic cells, we demonstrated that inducible NO synthase colocalizes with ERGIC/Golgi markers, notably Sec22b and its partner syntaxin 5, in the cytoplasm and at the phagosome. Pharmacological blockade of the secretory pathway hindered NO and cytokine release, and inhibited NF-κB translocation to the nucleus. Importantly, RNA interference-mediated silencing of Sec22b revealed that NO and cytokine production were abrogated at the protein and mRNA levels. This correlated with reduced nuclear translocation of NF-κB. We also found that Sec22b co-occurs with NF-κB in both the cytoplasm and nucleus, pointing to a role for this SNARE in the shuttling of NF-κB. Collectively, our data unveiled a novel function for the ERGIC/Golgi, and its resident SNARE Sec22b, in the production and release of inflammatory mediators.

Leishmania donovani Metacyclic Promastigotes Impair Phagosome Properties in Inflammatory Monocytes.

Leishmaniasis, a debilitating disease with clinical manifestations ranging from self-healing ulcers to life-threatening visceral pathologies, is caused by protozoan parasites of the Leishmania genus. These professional vacuolar pathogens are transmitted by infected sand flies to mammalian hosts as metacyclic promastigotes and are rapidly internalized by various phagocyte populations. Classical monocytes are among the first myeloid cells to migrate to infection sites. Recent evidence shows that recruitment of these cells contributes to parasite burden and the establishment of chronic disease. However, the nature of Leishmania-inflammatory monocyte interactions during the early stages of host infection has not been well investigated. Here, we aimed to assess the impact of Leishmania donovani metacyclic promastigotes on antimicrobial responses within these cells. Our data showed that inflammatory monocytes are readily colonized by L. donovani metacyclic promastigotes, while infection with Escherichia coli is efficiently cleared. Upon internalization, metacyclic promastigotes inhibited superoxide production at the parasitophorous vacuole (PV) through a mechanism involving exclusion of NADPH oxidase subunits gp91phox and p47phox from the PV membrane. Moreover, we observed that unlike phagosomes enclosing zymosan particles, vacuoles containing parasites acidify poorly. Interestingly, whereas the parasite surface coat virulence glycolipid lipophosphoglycan (LPG) was responsible for the inhibition of PV acidification, impairment of the NADPH oxidase assembly was independent of LPG and GP63. Collectively, these observations indicate that permissiveness of inflammatory monocytes to L. donovani may thus be related to the ability of this parasite to impair the microbicidal properties of phagosomes.

Translational profiling of macrophages infected with Leishmania donovani identifies mTOR- and eIF4A-sensitive immune-related transcripts.

The protozoan parasite Leishmania donovani (L. donovani) causes visceral leishmaniasis, a chronic infection which is fatal when untreated. Herein, we investigated whether in addition to altering transcription, L. donovani modulates host mRNA translation to establish a successful infection. Polysome-profiling revealed that one third of protein-coding mRNAs expressed in primary mouse macrophages are differentially translated upon infection with L. donovani promastigotes or amastigotes. Gene ontology analysis identified key biological processes enriched for translationally regulated mRNAs and were predicted to be either activated (e.g. chromatin remodeling and RNA metabolism) or inhibited (e.g. intracellular trafficking and antigen presentation) upon infection. Mechanistic in silico and biochemical analyses showed selective activation mTOR- and eIF4A-dependent mRNA translation, including transcripts encoding central regulators of mRNA turnover and inflammation (i.e. PABPC1, EIF2AK2, and TGF-ß). L. donovani survival within macrophages was favored under mTOR inhibition but was dampened by pharmacological blockade of eIF4A. Overall, this study uncovers a vast yet selective reprogramming of the host cell translational landscape early during L. donovani infection, and suggests that some of these changes are involved in host defense mechanisms while others are part of parasite-driven survival strategies. Further in vitro and in vivo investigation will shed light on the contribution of mTOR- and eIF4A-dependent translational programs to the outcome of visceral leishmaniasis.

Leishmania braziliensis: Strain-Specific Modulation of Phagosome Maturation.

Leishmania (Viannia) braziliensis is responsible for the largest number of American tegumentary leishmaniasis (ATL) in Brazil. ATL can present several clinical forms including typical (TL) and atypical (AL) cutaneous and mucocutaneous (ML) lesions. To identify parasite and host factors potentially associated with these diverse clinical manifestations, we first surveyed the expression of two virulence-associated glycoconjugates, lipophosphoglycan (LPG) and the metalloprotease GP63 by a panel of promastigotes of Leishmania braziliensis (L. braziliensis) strains isolated from patients with different clinical manifestations of ATL and from the sand fly vector. We observed a diversity of expression patterns for both LPG and GP63, which may be related to strain-specific polymorphisms. Interestingly, we noted that GP63 activity varies from strain to strain, including the ability to cleave host cell molecules. We next evaluated the ability of promastigotes from these L. braziliensis strains to modulate phagolysosome biogenesis in bone marrow-derived macrophages (BMM), by assessing phagosomal recruitment of the lysosome-associated membrane protein 1 (LAMP-1) and intraphagosomal acidification. Whereas, three out of six L. braziliensis strains impaired the phagosomal recruitment of LAMP-1, only the ML strain inhibited phagosome acidification to the same extent as the L. donovani strain that was used as a positive control. While decreased phagosomal recruitment of LAMP-1 correlated with higher LPG levels, decreased phagosomal acidification correlated with higher GP63 levels. Finally, we observed that the ability to infect and replicate within host cells did not fully correlate with the inhibition of phagosome maturation. Collectively, our results revealed a diversity of strain-specific phenotypes among L. braziliensis isolates, consistent with the high genetic diversity within Leishmania populations.

The host cell secretory pathway mediates the export of Leishmania virulence factors out of the parasitophorous vacuole.

To colonize phagocytes, Leishmania subverts microbicidal processes through components of its surface coat that include lipophosphoglycan and the GP63 metalloprotease. How these virulence glycoconjugates are shed, exit the parasitophorous vacuole (PV), and traffic within host cells is poorly understood. Here, we show that lipophosphoglycan and GP63 are released from the parasite surface following phagocytosis and redistribute to the endoplasmic reticulum (ER) of macrophages. Pharmacological disruption of the trafficking between the ER and the Golgi hindered the exit of these molecules from the PV and dampened the cleavage of host proteins by GP63. Silencing by RNA interference of the soluble N-ethylmaleimide-sensitive-factor attachment protein receptors Sec22b and syntaxin-5, which regulate ER-Golgi trafficking, identified these host proteins as components of the machinery that mediates the spreading of Leishmania effectors within host cells. Our findings unveil a mechanism whereby a vacuolar pathogen takes advantage of the host cell's secretory pathway to promote egress of virulence factors beyond the PV.

Leishmania infantum Lipophosphoglycan-Deficient Mutants: A Tool to Study Host Cell-Parasite Interplay.

Lipophosphoglycan (LPG) is the major surface glycoconjugate of metacyclic Leishmania promastigotes and is associated with virulence in various species of this parasite. Here, we generated a LPG-deficient mutant of Leishmania infantum, the foremost etiologic agent of visceral leishmaniasis in Brazil. The L. infantum LPG-deficient mutant (Δlpg1) was obtained by homologous recombination and complemented via episomal expression of LPG1 (Δlpg1 + LPG1). Deletion of LPG1 had no observable effect on parasite morphology or on the presence of subcellular organelles, such as lipid droplets. While both wild-type and add-back parasites reached late phase in axenic cultures, the growth of Δlpg1 parasites was delayed. Additionally, the deletion of LPG1 impaired the outcome of infection in murine bone marrow-derived macrophages. Although no significant differences were observed in parasite load after 4 h of infection, survival of Δlpg1 parasites was significantly reduced at 72 h post-infection. Interestingly, L. infantum LPG-deficient mutants induced a strong NF-κB-dependent activation of the inducible nitric oxide synthase (iNOS) promoter compared to wild type and Δlpg1 + LPG1 parasites. In conclusion, the L. infantum Δlpg1 mutant constitutes a powerful tool to investigate the role(s) played by LPG in host cell-parasite interactions.

Understanding TGEV-ETEC Coinfection through the Lens of Proteomics: A Tale of Porcine Diarrhea.

Porcine diarrhea and gastroenteritis are major causes of piglet mortality that result in devastating economic losses to the industry. A plethora of pathogens can cause these diseases, with the transmissible gastroenteritis virus (TGEV) and enterotoxigenic Escherichia coli K88 (ETEC) being two of the most salient. In the December 2017 issue of Proteomics Clinical Aplications, Xia and colleagues used comparative proteomics to shed light on how these microbes interact to cause severe disease . The authors discovered that TGEV induces an epithelial-mesenchymal transition-like phenotype that augments cell adhesion proteins mediating the attachment of ETEC to intestinal epithelial cells. Moreover, coinfection was found to modulate several host proteins that could bolster pathogen persistence. Importantly, the authors observed that ETEC suppresses the production of inflammatory cytokines induced by TGEV, which may in turn promote the long-term survival of both microbes.

Macrophages Tell the Non-Professionals What to Do.

Phagocytosis by professional and non-professional phagocytes plays a critical role in tissue homeostasis and the immune response. Using an airway inflammation model, Han et al. (2016) report in Nature that macrophages secrete IGF-1 to signal epithelial cells to stop ingesting apoptotic cells while increasing the uptake of anti-inflammatory macrophage-derived microvesicles.

Leishmania survival in the macrophage: where the ends justify the means.

Macrophages are cells of the immune system that mediate processes ranging from phagocytosis to tissue homeostasis. Leishmania has evolved ingenious ways to adapt to life in the macrophage. The GP63 metalloprotease, which disables key microbicidal pathways, has recently been found to disrupt processes ranging from antigen cross-presentation to nuclear pore dynamics. New studies have also revealed that Leishmania sabotages key metabolic and signaling pathways to fuel parasite growth. Leishmania has also been found to induce DNA methylation to turn off genes controlling microbicidal pathways. These novel findings highlight the multipronged attack employed by Leishmania to subvert macrophage function.

Macrophage cytokines: involvement in immunity and infectious diseases.

The evolution of macrophages has made them primordial for both development and immunity. Their functions range from the shaping of body plans to the ingestion and elimination of apoptotic cells and pathogens. Cytokines are small soluble proteins that confer instructions and mediate communication among immune and non-immune cells. A portfolio of cytokines is central to the role of macrophages as sentries of the innate immune system that mediate the transition from innate to adaptive immunity. In concert with other mediators, cytokines bias the fate of macrophages into a spectrum of inflammation-promoting "classically activated," to anti-inflammatory or "alternatively activated" macrophages. Deregulated cytokine secretion is implicated in several disease states ranging from chronic inflammation to allergy. Macrophages release cytokines via a series of beautifully orchestrated pathways that are spatiotemporally regulated. At the molecular level, these exocytic cytokine secretion pathways are coordinated by multi-protein complexes that guide cytokines from their point of synthesis to their ports of exit into the extracellular milieu. These trafficking proteins, many of which were discovered in yeast and commemorated in the 2013 Nobel Prize in Physiology or Medicine, coordinate the organelle fusion steps that are responsible for cytokine release. This review discusses the functions of cytokines secreted by macrophages, and summarizes what is known about their release mechanisms. This information will be used to delve into how selected pathogens subvert cytokine release for their own survival.

Leishmania promastigotes induce cytokine secretion in macrophages through the degradation of synaptotagmin XI.

Synaptotagmins (Syts) are type-I membrane proteins that regulate vesicle docking and fusion in processes such as exocytosis and phagocytosis. We recently discovered that Syt XI is a recycling endosome- and lysosome-associated protein that negatively regulates the secretion of TNF and IL-6. In this study, we show that Syt XI is directly degraded by the zinc metalloprotease GP63 and excluded from Leishmania parasitophorous vacuoles by the promastigotes surface glycolipid lipophosphoglycan. Infected macrophages were found to release TNF and IL-6 in a GP63-dependent manner. To demonstrate that cytokine release was dependent on GP63-mediated degradation of Syt XI, small interfering RNA-mediated knockdown of Syt XI before infection revealed that the effects of small interfering RNA knockdown and GP63 degradation were not cumulative. In mice, i.p. injection of GP63-expressing parasites led to an increase in TNF and IL-6 secretion and to an augmented influx of neutrophils and inflammatory monocytes to the inoculation site. Both of these cell types have been shown to be infection targets and aid in the establishment of infection. In sum, our data revealed that GP63 induces proinflammatory cytokine release and increases infiltration of inflammatory phagocytes. This study provides new insight on how Leishmania exploits the immune response to establish infection.

Leishmania dices away cholesterol for survival.

Host lipid alterations are centrally involved in Leishmania donovani infection, and infected patients exhibit hypocholesterolemia. In this issue of Cell Host & Microbe, Ghosh et al. (2013) show that the metalloprotease GP63 released by L. donovani in the liver cleaves DICER1, inhibiting miR-122 maturation, which regulates cholesterol metabolism. These events decrease serum cholesterol and promote parasite growth.

Synaptotagmin XI regulates phagocytosis and cytokine secretion in macrophages.

Synaptotagmins (Syts) are a group of type I membrane proteins that regulate vesicle docking and fusion in processes such as exocytosis and phagocytosis. All Syts possess a single transmembrane domain, and two conserved tandem Ca(2+)-binding C2 domains. However, Syts IV and XI possess a conserved serine in their C2A domain that precludes these Syts from binding Ca(2+) and phospholipids, and from mediating vesicle fusion. Given the importance of vesicular trafficking in macrophages, we investigated the role of Syt XI in cytokine secretion and phagocytosis. We demonstrated that Syt XI is expressed in murine macrophages, localized in recycling endosomes, lysosomes, and recruited to phagosomes. Syt XI had a direct effect on phagocytosis and on the secretion of TNF and IL-6. Whereas small interfering RNA-mediated knockdown of Syt XI potentiated secretion of these cytokines and particle uptake, overexpression of an Syt XI construct suppressed these processes. In addition, Syt XI knockdown led to decreased recruitment of gp91(phox) and lysosomal-associated membrane protein-1 to phagosomes, suggesting attenuated microbicidal activity. Remarkably, knockdown of Syt XI ensued in enhanced bacterial survival. Our data reveal a novel role for Syt XI as a regulator of cytokine secretion, particle uptake, and macrophage microbicidal activity.