Leishmania commandeers the host inflammatory response through neutrophils

Update Research Focus

Leishmania commandeers the host inflammatory response through neutrophils Ryan C. Jochim and Clarissa Teixeira Vector Molecular Biology Unit, Laboratory of Malaria and Vector Research, 12735 Twinbrook Parkway, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MA 20852, USA

Neutrophils are the first cells to migrate to the site of tissue damage. Recent work has addressed Leishmania survival and entry into macrophages through the infection of neutrophils that are recruited as a normal response to sandfly bites. New findings indicate that Leishmania is able to escape from neutrophils and ‘silently’ enter macrophages, a modification of the ‘Trojan horse’ model. Neutrophil depletion impaired disease progression, indicating an important role for neutrophils in leishmaniasis.

The Trojan horse model of Leishmania establishment When phlebotomine sandflies take a blood meal, the mouthparts lacerate the epidermis and produce a small pool of blood from which the female fly imbibes. Sandflies infected with the parasite Leishmania can regurgitate the virulent metacyclic promastigote form of the parasite into the skin and cause disease in susceptible hosts. Disease progression is believed to be due to ineffective activation (or no activation at all) of parasitized host macrophages. These cells are known to be the permanent residence for Leishmania amastigotes and were believed to become infected directly by metacyclic promastigotes through complement-based opsonic ingestion and lectin-lipophosphoglycan phagocytosis [1–3]. However, the inoculation of metacyclic promastigotes, via needle, has been shown to trigger the innate influx of neutrophils that phagocytose and harbor viable parasites [4]. The infected neutrophils gradually undergo apoptosis and are ingested by macrophages, highlighting another means by which macrophages are parasitized by Leishmania [5]. This was termed the ‘Trojan horse model’ to describe the ‘silent’ entry of Leishmania into the host macrophages. New evidence redefines Leishmania entry into macrophages Recently, Peters et al. [6] proposed an alternative model after intricately characterizing the important role that neutrophils have in the establishment of Leishmania infection. Using two-photon intravital microscopy (2P-IVM), they were able to temporally track enhanced green-fluorescent-protein-expressing neutrophils (eGFPhi) and macrophages (eGFPlo) and the interactions between these cells and Leishmania major parasites expressing red fluorescent protein (RFP). The model involved the deposition of L. major metacyclic promastigotes into the ear auricle skin Corresponding author: Jochim, R.C. ([email protected]).

of C57BL/6 mice by infected Phlebotomus duboscqi sandflies or needle injection. After mice were bitten with non-infected or RFP L. major-infected P. duboscqi sandflies, neutrophils swarmed rapidly to the bite site. The abundant neutrophils centralized around individual feeding sites in the dermis and filled the bite wound in the epidermis, forming a neutrophil ‘plug’. The depositing of RFP L. major by infected P. duboscqi did not result in a significant difference in the number of neutrophils and macrophages recruited to the bitten tissue when compared with uninfected bites. Neutrophils, being a short-lived cell of the innate immune system, are less abundant six days after sandfly bites, whereas macrophages persist. A slight, but not statistically significant, increase in the number of macrophages occurred in RFP L. major-infected P. duboscqi bites, in contrast to a slight decline in the number of macrophages that occurred in tissue bitten by uninfected sandflies. An image taken via 2P-IVM three hours after the transmission of RFP L. major depicts neutrophils that have phagocytosed the parasites. Video of the neutrophils swarming towards the site of bites by RFP L. majorinfected P. duboscqi shows parasites with rather sedate motility; however, RFP parasites can be seen outside of the neutrophil ‘plug’. This might indicate that the regurgitation of Leishmania by the sandfly is forceful enough to aid in the dispersion of the parasites in the dermis or that other factors are contributing to the spread of parasites. Peters et al. demonstrate that a comparable number of neutrophils are recruited, after 24 h, to the site of either the infected sandfly bite or the needle inoculation of L. major parasites. The neutrophil population was more persistent in tissue receiving L. major-infected sandfly bites than in tissue receiving needle inoculation of L. major. This indicates that needle injection of Leishmania parasites is not fully representative of what occurs during the transmission by sandflies. The majority of sandflies transmit a low dose of parasites while blood feeding, inoculating <600 promastigotes [7]. For the purpose of imaging the interactions between Leishmania and neutrophils, the rest of the experiments were conducted using needle inoculation of a larger number (104–106) of parasites. RFP-expressing L. major parasites were found primarily within neutrophils two hours post infection and were identified within neutrophils up to 16 h post infection. Tracking the eGFPhi cells using time-lapse video, Peters et al. depict the rapid extravasation of neutrophils to the site of inoculation. Neutrophils were seen rapidly engulfing parasites after migrating to the site of inoculation and then remaining 145

Update stationary. Although a large number of neutrophils that have internalized RFP L. major can remain in the skin, a portion of these neutrophils will transit to the draining lymph nodes. The presence of Leishmania-infected neutrophils in the lymph node could have a large impact on triggering the initial immune response, similar to what has been described in Toxoplasma gondii- and Mycobacterium bovis-infected neutrophils. These recent works provide evidence that infected neutrophils are able to swarm towards lymph nodes and dictate the outcome of the immune response, indicating new potential functions for these cells [8,9]. Macrophages in ear tissue were also seen with intracellular parasites in the first few hours after inoculation; however, few macrophages contained RFP L. major. Leishmania parasites were seen primarily within neutrophils early after needle inoculation, despite the dominant presence of macrophages. The proportion of neutrophils and macrophages containing RFP L. major changes during the first week of infection and undergoes a transition from neutrophils to macrophages. Meanwhile, the leukocyte population shifts; neutrophils decrease and there is a concomitant increase in macrophages and dendritic cells. Neutrophils, being the first line of cellular defense of the immune system, possess the vital role of killing the pathogens that are phagocytosed [10–12]. Peters et al. isolated RFPhi–eGFPhi cells – neutrophils infected with RFP L. major – and demonstrated that the internalized parasites are viable and can cause disease in a naı¨ve mouse model. This corroborates previous in vitro and in vivo experiments showing the prompt phagocytosis and intracellular survival of Leishmania parasites within neutrophils, despite the microbicidal arsenal within neutrophils [4,5,13,14]. Perhaps the most notable finding reported by Peters et al. is the effect of depleting neutrophils in naı¨ve mice that are then infected by P. duboscqi bites. Mice depleted of neutrophils had a significantly reduced incidence of infection and number of parasites present in the ear four weeks after infection. Interestingly, depletion of neutrophils did not lead to an increase in the uptake of parasites by macrophages and dendritic cells, reinforcing the niche that neutrophils might provide for parasite survival. Parasites surviving within apoptotic neutrophils are a defining factor in the Trojan horse theory of Leishmaniainfected neutrophils ‘silently’ entering macrophages. The paper by Peters et al. was the first report of apoptotic neutrophils releasing Leishmania parasites that are then ingested by other phagocytic cells. We emphasize here that this finding amends the Trojan horse model based on the previous observation of macrophage uptake of apoptotic, infected neutrophils [5]. Peters et al. propose two hypotheses for the early Leishmania–neutrophil interactions. The first hypothesis proposes that the neutrophil is a site of temporary refuge for the parasite, and the second hypothesis proposes that the neutrophil is a site of adaptation for Leishmania to better parasitize macrophages. Both hypotheses are intriguing and they could be occurring simultaneously, better explaining how Leishmania has adapted to use the natural host response to survive and establish successfully. 146

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An additional hypothesis proposed by Peters et al. is that the macrophages are responding to the natural inflammatory response by ingesting apoptotic neutrophils and eliciting a well-known anti-inflammatory effect. In support of this hypothesis, neutrophil-depleted mice that were bitten by L. major-infected P. duboscqi showed an increase in interleukin (IL)-1a and IL-1b production in the skin. In control mice, the recruitment and presumed apoptosis of neutrophils within the surrounding tissue resulted in decreased IL-1 production. Both IL-1a and IL-1b are linked with the inflammatory response; however, vital roles have also been described for these cytokines because they are able to activate T cells [15,16]. This indicates that Leishmania parasites infecting the recruited neutrophils cause a downregulation of IL-1 production that would abrogate T-cell activation and result in subsequent disease. Neutrophils – a new focus for future research The work by Peters et al. elegantly details the interaction of neutrophils with L. major using the natural infection by P. duboscqi bites and provides an amended model of the Trojan horse route of Leishmania establishment. This reaffirms the importance of neutrophils as primary host cells playing an essential part in early disease development. New directions should follow the impact of neutrophils and dendritic cells, keratinocytes, endothelial cells, and fibroblasts present at the skin in disease maintenance and in the establishment of an adaptive response [17–19]. In addition to neutrophils, several other factors, such as sandfly saliva and biting behavior of infected flies, are likely to modulate the inflammatory response and influence disease outcome [20–22]. The Leishmaniaderived promastigote secretory gel and promastigote chemotactic factor should also be considered important immunomodulators [23,24]. Future research should focus on addressing the role of these factors in the context of Leishmania exploiting the natural host response to sandfly bites. Acknowledgements This work was supported by the Intramural Research Program of the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health.

References 1 Wozencraft, A.O. and Blackwell, J.M. (1987) Increased infectivity of stationary-phase promastigotes of Leishmania donovani: correlation with enhanced C3 binding capacity and CR3-mediated attachment to host macrophages. Immunology 60, 559–563 2 Mosser, D.M. and Edelson, P.J. (1987) The third component of complement (C3) is responsible for the intracellular survival of Leishmania major. Nature 327, 329–331 3 Wilson, M.E. and Pearson, R.D. (1988) Roles of CR3 and mannose receptors in the attachment and ingestion of Leishmania donovani by human mononuclear phagocytes. Infect. Immun. 56, 363–369 4 Laufs, H. et al. (2002) Intracellular survival of Leishmania major in neutrophil granulocytes after uptake in the absence of heat-labile serum factors. Infect. Immun. 70, 826–835 5 van Zandbergen, G. et al. (2004) Cutting edge: neutrophil granulocyte serves as a vector for Leishmania entry into macrophages. J. Immunol. 173, 6521–6525 6 Peters, N.C. et al. (2008) In vivo imaging reveals an essential role for neutrophils in leishmaniasis transmitted by sand flies. Science 321, 970–974

Update 7 Kimblin, N. et al. (2008) Quantification of the infectious dose of Leishmania major transmitted to the skin by single sand flies. Proc. Natl. Acad. Sci. U. S. A. 105, 10125–10130 8 Abadie, V. et al. (2005) Neutrophils rapidly migrate via lymphatics after Mycobacterium bovis BCG intradermal vaccination and shuttle live bacilli to the draining lymph nodes. Blood 106, 1843–1850 9 Chtanova, T. et al. (2008) Dynamics of neutrophil migration in lymph nodes during infection. Immunity 29, 487–496 10 Nauseef, W.M. (2007) How human neutrophils kill and degrade microbes: an integrated view. Immunol. Rev. 219, 88–102 11 Segal, A.W. (2005) How neutrophils kill microbes. Annu. Rev. Immunol. 23, 197–223 12 Roos, D. et al. (2003) Oxidative killing of microbes by neutrophils. Microbes Infect. 5, 1307–1315 13 Muller, K. et al. (2001) Chemokines, natural killer cells and granulocytes in the early course of Leishmania major infection in mice. Med. Microbiol. Immunol. (Berl.) 190, 73–76 14 Gueirard, P. et al. (2008) Trafficking of Leishmania donovani promastigotes in non-lytic compartments in neutrophils enables the subsequent transfer of parasites to macrophages. Cell. Microbiol. 10, 100–111 15 Nambu, A. et al. (2006) IL-1b, but not IL-1a, is required for antigenspecific T cell activation and the induction of local inflammation in the delayed-type hypersensitivity responses. Int. Immunol. 18, 701–712 16 Nakae, S. et al. (2001) IL-1 alpha, but not IL-1 beta, is required for contact-allergen-specific T cell activation during the

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1471-4922/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.pt.2009.01.001 Available online 5 March 2009

Research Focus

A novel twist to protein secretion in eukaryotes Richard Zimmermann1 and Gregory L. Blatch2 1

Medical Biochemistry and Molecular Biology, Saarland University, 66421 Homburg, Germany Biomedical Biotechnology Research Unit, Department of Biochemistry, Microbiology and Biotechnology, Rhodes University, Grahamstown 6140, South Africa

2

A recent functional analysis of the protein translocase, which is present in the trypanosomal endoplasmic reticulum membrane, by Michaeli and co-workers has indicated an unexpected diversity in the mechanisms and components of protein secretion in eukaryotes and might eventually pave the way for the development of anti-trypanosomal drugs. Furthermore, the work on these human parasites also supports conclusions that were drawn previously for components of protein secretion in human cells on the basis of in vitro studies.

Protein secretion in mammals, yeast and bacteria Protein transport into the endoplasmic reticulum (ER) is the first step in the biogenesis of most extracellular and plasma membrane proteins in eukaryotic cells [1,2]. Typically, it involves N-terminal signal peptides in the precursor proteins and a sophisticated transport machinery (Figure 1). Two mechanisms (termed ‘co-translational mechanism’ and ‘post-translational mechanism’) can be distinguished; they differ in their relationship to translation and with respect to the relevant cytosolic components (signal-recognition particle, or SRP, versus Corresponding author: Zimmermann, R. ([email protected]).

cognate heat-shock protein 70, or Hsc70, respectively). The two mechanisms merge at the ER membrane, specifically at the Sec61 complex that comprises a-, b- and g-subunits and forms a gated polypeptide-conducting channel [3,4]. Transport can be divided into three stages: targeting, membrane insertion and completion of translocation. In yeast and mammalian cells, targeting or specific membrane association in co-translational transport involves SRP and its receptor on the ER surface (SRP receptor, or SR) [5,6]. In mammalian cells, this pathway is the predominant one, whereas in yeast, SRP-dependent (co-translational) and SRP-independent (post-translational) pathways are equally important. In yeast, targeting in post-translational transport of precursor proteins involves a heterotrimeric complex of membrane proteins – comprising Sec62p, Sec71p (also termed ‘Sec66p’) and Sec72p (also termed ‘Sec67p’) – that serves as a signal peptide receptor [7,8] (Table 1). This pathway is used predominantly by precursors with less hydrophobic signal peptides [9]. In yeast, as well as in mammals, membrane insertion and completion of translocation occur at the level of the Sec61 complex and involve additional components, such as the ER-lumenal chaperone immunoglobulin heavy-chain-binding protein (BiP) and its membrane receptor and co-chaperone Sec63 [10–15]. Based on in vitro 147