Maximiliano G. Gutierrez

Neutrophils sense microbe size and selectively release neutrophil extracellular traps in response to large pathogens

Neutrophils are critical for antifungal defense, but the mechanisms that clear hyphae and other pathogens that are too large to be phagocytosed remain unknown. We found that neutrophils sensed microbe size and selectively released neutrophil extracellular traps (NETs) in response to large pathogens, such as Candida albicans hyphae and extracellular aggregates of Mycobacterium bovis, but not in response to small yeast or single bacteria. NETs were fundamental in countering large pathogens in vivo. Phagocytosis via dectin-1 acted as a sensor of microbe size and prevented NET release by downregulating the translocation of neutrophil elastase (NE) to the nucleus. Dectin-1 deficiency led to aberrant NET release and NET-mediated tissue damage during infection. Size-tailored neutrophil responses cleared large microbes and minimized pathology when microbes were small enough to be phagocytosed.

Coxiella burnetii Localizes in a Rab7-Labeled Compartment with Autophagic Characteristics

ABSTRACT The obligate intracellular bacterium Coxiella burnetii, the agent of Q fever in humans and of coxiellosis in other animals, survives and replicates within large, acidified, phagolysosome-like vacuoles known to fuse homo- and heterotypically with other vesicles. To further characterize these vacuoles, HeLa cells were infected with C. burnetii phase II; 48 h later, bacteria-containing vacuoles were labeled by LysoTracker, a marker of acidic compartments, and accumulated monodansylcadaverine and displayed protein LC3, both markers of autophagic vacuoles. Furthermore, 3-methyladenine and wortmannin, agents known to inhibit early stages in the autophagic process, each blocked Coxiella vacuole formation. These autophagosomal features suggest that Coxiella vacuoles interact with the autophagic pathway. The localization and role of wild-type and mutated Rab5 and Rab7, markers of early and late endosomes, respectively, were also examined to determine the role of these small GTPases in the trafficking of C. burnetii phase II. Green fluorescent protein (GFP)-Rab5 and GFP-Rab7 constructs were overexpressed and visualized by fluorescence microscopy. Coxiella-containing large vacuoles were labeled with wild-type Rab7 (Rab7wt) and with GTPase-deficient mutant Rab7Q67L, whereas no colocalization was observed with the dominant-negative mutant Rab7T22N. The vacuoles were also decorated by GFP-Rab5Q79L but not by GFP-Rab5wt. These results suggest that Rab7 participates in the biogenesis of the parasitophorous vacuoles.

Protective role of autophagy against Vibrio cholerae cytolysin, a pore-forming toxin from V. cholerae

Autophagy is the unique, regulated mechanism for the degradation of organelles. This intracellular process acts as a prosurvival pathway during cell starvation or stress and is also involved in cellular response against specific bacterial infections. Vibrio cholerae is a noninvasive intestinal pathogen that has been studied extensively as the causative agent of the human disease cholera. V. cholerae illness is produced primarily through the expression of a potent toxin (cholera toxin) within the human intestine. Besides cholera toxin, this bacterium secretes a hemolytic exotoxin termed V. cholerae cytolysin (VCC) that causes extensive vacuolation in epithelial cells. In this work, we explored the relationship between the vacuolation caused by VCC and the autophagic pathway. Treatment of cells with VCC increased the punctate distribution of LC3, a feature indicative of autophagosome formation. Moreover, VCC-induced vacuoles colocalized with LC3 in several cell lines, including human intestinal Caco-2 cells, indicating the interaction of the large vacuoles with autophagic vesicles. Electron microscopy analysis confirmed that the vacuoles caused by VCC presented hallmarks of autophagosomes. Additionally, biochemical evidence demonstrated the degradative nature of the VCC-generated vacuoles. Interestingly, autophagy inhibition resulted in decreased survival of Caco-2 cells upon VCC intoxication. Also, VCC failed to induce vacuolization in Atg5−/− cells, and the survival response of these cells against the toxin was dramatically impaired. These results demonstrate that autophagy acts as a cellular defense pathway against secreted bacterial toxins.

NF-κB Activation Controls Phagolysosome Fusion-Mediated Killing of Mycobacteria by Macrophages

Macrophages can potentially kill all mycobacteria by poorly understood mechanisms. In this study, we explore the role of NF-κB in the innate immune response of macrophages against Mycobacterium smegmatis, a nonpathogenic mycobacterium efficiently killed by macrophages, and Mycobacterium avium which survives within macrophages. We show that infection of macrophages with M. smegmatis induces an activation of NF-κB that is essential for maturation of mycobacterial phagosomes and bacterial killing. In contrast, the pathogenic M. avium partially represses NF-κB activation. Using microarray analysis, we identified many lysosomal enzymes and membrane-trafficking regulators, including cathepsins, LAMP-2 and Rab34, were regulated by NF-κB during infection. Our results argue that NF-κB activation increases the synthesis of membrane trafficking molecules, which may be rate limiting for regulating phagolysosome fusion during infection. The direct consequence of NF-κB inhibition is the impaired delivery of lysosomal enzymes to M. smegmatis phagosomes and reduced killing. Thus, the established role of NF-κB in the innate immune response can now be expanded to include regulation of membrane trafficking during infection.

Golgi-to-phagosome transport of acid sphingomyelinase and prosaposin is mediated by sortilin

Sortilin, also known as neurotensin receptor 3 (NTR3), is a transmembrane protein with a dual function. It acts as a receptor for neuromediators and growth factors at the plasma membrane, but it has also been implicated in binding and transport of some lysosomal proteins. However, the role of sortilin during phagosome maturation has not been investigated before. Here, we show that in macrophages, sortilin is mainly localized in the Golgi and transported to latex-bead phagosomes (LBPs). Using live-cell imaging and electron microscopy, we found that sortilin is delivered to LBPs in a manner that depends on its cytoplasmic tail. We also show that sortilin participates in the direct delivery of acid sphingomyelinase (ASM) and prosaposin (PS) to the phagosome, bypassing fusion with lysosomal compartments. Further analysis confirmed that ASM and PS are targeted to the phagosome by sortilin in a Brefeldin-A-sensitive pathway. Analysis of primary macrophages isolated from Sort1−/− mice indicated that the delivery of ASM and PS, but not pro-cathepsin D, to LBPs was severely impaired. We propose a pathway mediated by sortilin by which selected lysosomal proteins are transported to the phagosome along a Golgi-dependent route during the maturation of phagosomes.

Role of lipids in killing mycobacteria by macrophages: evidence for NF‐κB‐dependent and ‐independent killing induced by different lipids

We have shown that several lipids can modulate the macrophage innate immune response against mycobacteria and enhance their killing. Since NF‐κB is required for mycobacterial killing, we tested the ability of lipids to activate NF‐κB in uninfected macrophages and those infected with mycobacteria. In uninfected cells, sphingomyelin (SM), phosphatidylinositol‐4‐phosphate (PIP) and arachidonic acid (AA) enhanced NF‐κB activation and the cell surface expression of CD69, a macrophage activation marker regulated by NF‐κB. Sphingosine (Sph), sphingosine‐1‐phosphate (S1P), diacylglycerol (DAG), eicosapentanoic acid (EPA) and phosphatidyl choline (PC) failed to activate either NF‐κB or CD69. Ceramide (Cer) activated CD69 expression without activating NF‐κB. In Mycobacterium smegmatis‐infected cells, NF‐κB was transiently activated in a manner that was enhanced by SM, PIP and AA. In contrast Mycobacterium avium mostly repressed NF‐κB activation and only SM and AA could induce its partial activation. While lipids that activate NF‐κB in uninfected cells tend to kill mycobacteria in macrophages Sph and S1P failed to activate NF‐κB under most conditions but nevertheless enhanced killing of M. smegmatis, M. avium and M. tuberculosis H37Rv. Our results argue that both NF‐κB‐dependent and ‐independent mechanisms are involved in macrophage killing of mycobacteria and that both mechanisms can be enhanced by selected lipids.

Functional role(s) of phagosomal Rab GTPases.

Rab GTPases are at the central node of the machinery that regulates trafficking of organelles, including phagosomes. Thanks to the unique combination of high quality phagosome purification with highly sensitive proteomic studies, the network of Rab proteins that are dynamically associated with phagosomes during the process of maturation of this organelle is relatively well known. Whereas the phagosomal functions of many of the Rab proteins associated with phagosomes are characterized, the role(s) of most of these trafficking regulators remains to be identified. In some cases, even when the function in the context of phagosome biology is described, phagosomal Rab proteins seem to have similar roles. This review summarizes the current knowledge about the identity and function of phagosomal Rab GTPases, with a particular emphasis on new evidence that clarify these seemingly overlapping Rab functions during phagosome maturation.

The Two Faces of Autophagy: Coxiella and Mycobacterium

In the world of pathogen-host cell interactions, the autophagic pathway has beenrecently described as a component of the innate immune response against intracellularmicroorganisms. Indeed, some bacterial survival mechanisms are hampered when thisprocess is activated. Mycobacterium tuberculosis infection of macrophages, for example,is impaired upon autophagy induction and the bacterial phagosomes are redirected toautophagosomes. On the other hand, pathogens like Coxiella burnetii are benefited bythis cellular response and subvert the autophagy process resulting in a more efficient replication.We study at the molecular level these two different faces of the autophagy processin pathogen life in order to elucidate the intricate routes modulated by the microorganismsas survival strategies.

Size-dependent mechanism of cargo sorting during lysosome-phagosome fusion is controlled by Rab34

Phagosome maturation is an essential part of the innate and adaptive immune response. Although it is well established that several Ras-related proteins in brain (Rab) proteins become associated to phagosomes, little is known about how these phagosomal Rab proteins influence phagosome maturation. Here, we show a specific role for Rab34 and mammalian uncoordinated 13-2 (Munc13-2) in phagolysosome biogenesis and cargo delivery. Rab34 knockdown impaired the fusion of phagosomes with late endosomes/lysosomes and high levels of active Rab34 promoted this process. We demonstrate that Rab34 enhances phagosome maturation independently of Rab7 and coordinates phagolysosome biogenesis through size-selective transfer of late endosomal/lysosomal cargo into phagosomes. More importantly, we show that Rab34 mediates phagosome maturation through the recruitment of the protein Munc13-2. Finally, we report that the alternative maturation pathway controlled by Rab34 is critical for mycobacterial killing because Rab34 silencing resulted in mycobacterial survival, and Rab34 expression led to mycobacterial killing. Altogether, our studies uncover Rab34/Munc13-2 as a critical part of an alternative Rab7-independent phagosome maturation machinery and lysosome-mediated killing of mycobacteria.

Lymph node-derived lymphatic endothelial cells express functional costimulatory molecules and impair dendritic cell-induced allogenic T-cell proliferation

Lymphatic endothelial cells (LECs) interact with different immune cells, including T cells within lymph nodes (LNs). However, direct interactions of LECs with immune cells have yet to be investigated. In vitro studies were performed to characterize primary cultures of human LECs derived from LNs in their capacity of interacting with T cells. The results show that LECs express HLA molecules and functional costimulatory molecules needed for T‐cell activation. A direct binding of LECs and T cells was detected in cell cultures connected with a clustering of costimulatory molecules on the contact phase. LECs were also able to take up and process antigens. However, major histocompatibility complex class II+ LECs fail to induce allogeneic T‐cell proliferation. Interestingly, supernatants of IFN‐γ activated LECs impair proliferation of T cells cocultured with allogeneic dendritic cells, suggesting an inhibitory role of LECs. Indoleamine 2,3 dioxygenase was identified as one inhibitory molecule, which may be responsible for the impaired CD4+ T‐cell proliferation. Our observations suggest a regulatory function for activated LECs on CD4+ T cells, which may play a role in vivo in the maintenance of the critical balance between tolerance and recall responses.—Nörder, M., Gutierrez, M. G., Zicari, S., Cervi, E., Caruso, A., Guzmán, C. A. Lymph node‐derived lymphatic endothelial cells express functional costimulatory molecules and impair dendritic cell‐induced allogenic T‐cell proliferation. FASEB J. 26, 2835–2846 (2012). www.fasebj.org