Apoptosis driven infection

Professional phagocytes like polymorphonuclear neutrophil granulocytes (PMN) and macrophages (MF) kill pathogens as the first line of defense. These cells possess numerous effector mechanisms to eliminate a threat at first contact. However, several microorganisms still manage to evade phagocytic killing, survive and retain infectivity. Some pathogens have developed strategies to silently infect their preferred host phagocytes. The best example of an immune silencing phagocytosis process is the uptake of apoptotic cells. Immune responses are suppressed by the recognition of phosphatidylserine (PS) on the outer leaflet of their plasma membrane. Taking Leishmania major as a prototypic intracellular pathogen, we showed that these organisms can use the apoptotic "eat me" signal PS to silently enter PMN. PS-positive and apoptotic parasites, in an altruistic way, enable the intracellular survival of the viable parasites. Subsequently these pathogens again use PS exposition, now on infected PMN, to silently invade their definitive host cells, the MF. In this review, we will focus on L. major evasion strategies and discuss other pathogens and their use of the apoptotic "eat me" signal PS to establish infection.     

Attraction of phagocytes by apoptotic cells is mediated by lysophosphatidylcholine

Apoptotic cells promote the phagocytosis of themselves via the exposure of “eat me” signals. Furthermore, a second, “find me” signal is required to attract phagocytes. In this review the double function of lysophosphatidylcholine for the clearence of apoptotic material as factor leading to attraction of phagocytes as a soluble “find me” signal and opsonisation of apoptotic cells as a membrane bound “eat me” signal is outlined. Lysophosphatidylcholine, exerting this bivalent function, leads to a sufficient clearance of apoptotic cell as a “keep calm” mechanism to prevent activation of the immune system by secondary necrosis.     

Attraction of phagocytes by apoptotic cells is mediated by lysophosphatidylcholine

Apoptotic cells promote the phagocytosis of themselves via the exposure of "eat me" signals. Furthermore, a second, "find me" signal is required to attract phagocytes. In this review the double function of lysophosphatidylcholine for the clearance of apoptotic material as factor leading to attraction of phagocytes as a soluble "find me" signal and opsonisation of apoptotic cells as a membrane bound "eat me" signal is outlined. Lysophosphatidylcholine, exerting this bivalent function, leads to a sufficient clearance of apoptotic cell as a "keep calm" mechanism to prevent activation of the immune system by secondary necrosis.     

Plasma membrane alterations during apoptosis: role in corpse clearance

Apoptosis is a program of cellular self-destruction culminating in the clearance of cell corpses by neighboring macrophages. Studies in recent years have served to characterize a number of structural and molecular plasma membrane alterations that act in concert to mediate efficient engulfment of cell corpses. Hence, "eat me" signals, including the anionic phospholipid phosphatidylserine (PS) and its oxidized counterpart, PS-OX, as well as the PS-binding protein, annexin I, are exposed on the surface of effete cells and function to mediate engulfment by neighboring phagocytic cells. Plasma membrane blebbing (zeiosis), a common feature of the apoptotic program, provides a structural context for the exposition of recognition signals insofar as PS molecules aggregate on the surface of these membrane protrusions. Apoptotic cells also secrete chemotactic factors ("seek me" signals), such as the phospholipid lysophosphatidylcholine, that recruit phagocytes to the site of the apoptotic lesion. Taken together, these events serve to mediate the disposal of effete cells prior to their necrotic disintegration, thus preventing the inflammation and tissue scarring that would otherwise ensue.     

Phagosome proteomics to study Leishmania’s intracellular niche in macrophages

Intracellular pathogens invade their host cells and replicate within specialized compartments. In turn, the host cell initiates a defensive response trying to kill the invasive agent. As a consequence, intracellular lifestyle implies morphological and physiological changes in both pathogen and host cell. Leishmania spp. are medically important intracellular protozoan parasites that are internalized by professional phagocytes such as macrophages, and reside within the parasitophorous vacuole inhibiting their microbicidal activity. Whereas the proteome of the extracellular promastigote form and the intracellular amastigote form have been extensively studied, the constituents of Leishmania’s intracellular niche, an endolysosomal compartment, are not fully deciphered. In this review we discuss protocols to purify such compartments by means of an illustrating example to highlight generally relevant considerations and innovative aspects that allow purification of not only the intracellular parasites but also the phagosomes that harbor them and analyze the latter by gel free proteomics.     

The Influence of Different Cultivating Conditions on Polymorphonuclear Leukocyte Apoptotic Process In Vitro,II: Ultrastructural Characteristics of PMN Populations Incubated with Proteinase 3 Anti-neutrophil Autoantibodies

The present study shows the effects of proteinase 3 anti-neutrophil cytoplasmic autoantibodies (PR3 ANCA) on polymorphonuclear leukocytes (PMN) apoptotic processes in vitro. The results are part of a generalized morphological analysis of 3 identical experiments on the influence of different cultivating conditions on the apoptotic processes. As controls, the authors use the results on spontaneous PMN apoptosis (Guejes L, Zurgil N, Deutsch M, Gilburd B, Shoenfeld Y. Ultrastruct Pathol. 2003;27:23–32) and PMN populations incubated with normal human IgG. Interaction of PR3 ANCA with the target antigen proteinase 3 (PR3) is one of the crucial pathogenic factors in Wegener granulomatosis (systemic autoimmune vasculitis). Following 40 min and 12 h incubation, PMN populations were evaluated by light microscopy, transmission electron microscopy, and immunogold electron microscopy. Twelve-hour cultures, either control or incubated with PR3 ANCA, contained different cell forms ranging from normal cells to cells at the final stages of apoptosis. Neutrophils at the state of complete manifestation of apoptotic phenotype were analyzed and compared. Three morphologically distinct apoptotic cell lines were characteristic for all PMN populations studied, regardless of cultivating conditions. As in spontaneous apoptosis, these cell lines are code-named “first,” “second,” and “third.” The present study has shown, firstly, that in the presence of PR3 ANCA, all 3 apoptotic lines were modified or altered. Secondly, the modifications or alterations of apoptotic cell lines effected by PR3 ANCA are specific for each cell line: the “first” line is characterized by intensification and modification of activation; the “second” by vacuolized cell forms; and the “third” by pronounced lytic alterations of the nuclei, while the cytoplasm is fully identical to that of control cell lines.     

Exogenous Calreticulin,incorporated onto non-infective Trypanosoma cruzi epimastigotes,promotes their internalization into mammal host cells

Chagas disease is an endemic pathology in Latin America, now emerging in developed countries, caused by the intracellular protozoan Trypanosoma cruzi, whose life cycle involves three stages: amastigotes, epimastigotes, and trypomastigotes. T. cruzi Calreticulin (TcCRT), an endoplasmic reticulum resident chaperone, translocates to the external cellular membrane, where it captures complement component C1, ficolins and MBL, thus inactivating the classical and lectin pathways. Trypomastigote-bound C1 is detected as an “eat me” signal by macrophages and promotes the infective process. Unlike infective trypomastigotes, non-infective epimastigotes either do not express or express only marginal levels of TcCRT on their external membrane. We show that epimastigotes bind exogenous rTcCRT to their cellular membrane and, in the presence of C1q, this parasite form is internalized into normal fibroblasts. On the other hand, Calreticulin (CRT)-deficient fibroblasts show impaired parasite internalization. In synthesis, CRT from both parasite and host cell origin is important in the establishment of C1q-dependent first contacts between parasites and host cells.     

Moonlighting osteoclasts as undertakers of apoptotic cells

Rapid clearance of apoptotic cells, frequently referred to as efferocytosis, is crucial for the maintenance of tissue homeostasis and the prevention of autoimmunity. The common model of apoptotic cell clearance involves a system of released “Find me” and exposed “Eat me” signals on apoptotic cells, detected and recognized by matching receptors on macrophages or dendritic cells (DC), referred to as the phagocytic synapse. Osteoclasts share the monocyte lineage with these professional mononuclear phagocytes, thus raising the question if, in addition to bone resorption, osteoclasts can act as scavengers for apoptotic cells. Our qPCR data clearly show that osteoclasts express most of the genes required for dying cell clearance at mRNA levels similar to or even higher than those observed in M1-macrophages, M2-macrophages or DC. Our microscopical analyses reveal that osteoclasts in fact can bind and/or engulf apoptotic cells in an essentially serum-independent fashion. Together with our data on the abundance of the respective mRNAs, these results identify the vitronectin receptor (integrin α_νβ₃)/milk fat globule-EGF factor 8 protein (MFG-E8) axis, the scavenger receptors (CD36, CD68 and class A macrophage scavenger receptor (SR-A)), the complement/complement receptor axis, the Mer/Tyro3/Protein S axis, and the phosphatidylserine (PS) receptor brain-specific angiogenesis inhibitor 1 (BAI1) as the most promising candidates to be involved in osteoclast-mediated efferocytosis.     

The inhibitory receptor CD300a is essential for neutrophil-mediated clearance of urinary tract infection in mice

Neutrophils play a crucial role in immune defense against and clearance of uropathogenic Escherichia coli (UPEC)-mediated urinary tract infection, the most common bacterial infection in healthy humans. CD300a is an inhibitory receptor that binds phosphatidylserine and phosphatidylethanolamine, presented on the membranes of apoptotic cells. CD300a binding to phosphatidylserine and phosphatidylethanolamine, also known as the “eat me” signal, mediates immune tolerance to dying cells. Here, we demonstrate for the first time that CD300a plays an important role in the neutrophil-mediated immune response to UPEC-induced urinary tract infection. We show that CD300a-deficient neutrophils have impaired phagocytic abilities and despite their increased accumulation at the site of infection, they are unable to reduce bacterial burden in the bladder, which results in significant exacerbation of infection and worse host outcome. Finally, we demonstrate that UPEC's pore forming toxin α-hemolysin induces upregulation of the CD300a ligand on infected bladder epithelial cells, signaling to neutrophils to be cleared.     

Neutrophil granulocytes as host cells and transport vehicles for intracellular pathogens: apoptosis as infection-promoting factor

Polymorphonuclear neutrophil granulocytes (PMN) are primary antimicrobial effector cells of the innate immune system and serve to destroy invading pathogens. Although most ingested microorganisms are killed readily inside PMN, several obligate or facultative intracellular pathogens survive even in this hostile environment. Extension of the life span of neutrophils is a general escape mechanism of pathogens residing in PMN. However, after 2-4 days, even infected neutrophils become apoptotic and are phagocytosed by macrophages. Since microbes entering macrophages via the uptake of infected apoptotic PMN may survive and multiply in macrophages, apoptotic neutrophils can serve as "Trojan horses" for certain pathogens. Interfering with activating signaling pathways appears to be another potent mechanism by which intracellular microorganisms suppress cellular activation in neutrophils. In addition to provide a short overview of the topic, the present review aims to summarize our own findings regarding the interaction between human neutrophils and intracellular pathogens as well as regarding the disease promoting role of apoptotic cells after infection with Leishmania major.     

Unconventional apoptosis of polymorphonuclear neutrophils (PMN): staurosporine delays exposure of phosphatidylserine and prevents phagocytosis by MΦ-2 macrophages of PMN

Apoptosis of polymorphonuclear neutrophils (PMN) and subsequent ‘silent’ removal represents an important check-point for the resolution of inflammation. Failure in PMN clearance resulting in secondary necrosis-driven tissue damage has been implicated in conditions of chronic inflammation and autoimmunity. Apoptotic PMN undergo profound biophysical changes that warrant their efficient recognition and uptake by phagocytes before fading to secondary necrosis. In this study, we demonstrate that staurosporine (STS), a non-selective but potent inhibitor of cyclin-dependent kinase and protein kinase C, exerts a drastic impact on PMN apoptosis. PMN treated with STS underwent an unconventional form of cell death characterized by a delayed exposure of aminophospholipids, including phosphatidylserine (PS) and phosphatidylethanolamine and an increased exposure of neo-glycans. STS caused an impaired cellular fragmentation and accelerated DNA fragmentation. Phagocytosis of STS-treated PMN lacking PS on their surfaces was decreased significantly, which highlights the importance of PS for the clearance of apoptotic PMN. Specific opsonization with immune complexes completely restored phagocytosis of STS-treated PMN, demonstrating the efficiency of back-up clearance pathways in the absence of PS exposure.     

"Eat me" and "don't eat me" signals govern the innate immune response and tissue repair in the CNS: emphasis on the critical role of the complement system

A full innate immune system (e.g. complement system, scavenger receptors, Toll-like receptors (TLR)) has been described in the CNS and is thought to be an extremely efficient army designed to fight against invading pathogens and toxic cell debris such as apoptotic cells and amyloid fibrils. The binding of soluble or secreted innate immune molecules on pathogen-associated molecular patterns (PAMPs) as well as apoptotic cell-associated molecular patterns (ACAMPs) provide several "eat me" signals to promote the safe disposal of the intruders by professional and amateur phagocytes. These patterns are deciphered by receptors (pattern recognition receptors, PRRs; e.g. CR3) that control phagocytosis and associated inflammatory response depending on the meaning of these signals. Importantly, in order to avoid excessive collateral damage of surrounding cells, it is increasingly evident that "don't eat me" signals (coined herein as self-associated molecular patterns, SAMPs; e.g. complement regulatory proteins, CD200) are of paramount importance to signal a robust anti-inflammatory response and promote tissue repair. Further knowledge of the innate immune response in the CNS will greatly help to delineate the novel therapeutic routes to protect from CNS inflammation and neurodegeneration.     

Afa/Dr-Expressing,Diffusely Adhering Escherichia coli Strain C1845 Triggers F1845 Fimbria-Dependent Phosphatidylserine Externalization on Neutrophil-Like Differentiated PLB-985 Cells through an Apoptosis-Independent Mechanism

The enterovirulent Escherichia coli strains potentially involved in inflammatory bowel diseases include diffusely adherent strains expressing Afa/Dr fimbriae (Afa/Dr DAEC). We have previously observed type 1 pilus-mediated interleukin-8 (IL-8) hyperproduction in infected neutrophils. As pathogen induction of host cell death programs and clearance of apoptotic infected cells are crucial for innate immune system homeostasis and host integrity, we examined modulation of neutrophil cell death by Afa/Dr DAEC. Using the human PLB-985 cell line differentiated into fully mature neutrophils, we found that the wild-type enterovirulent E. coli strain C1845 and the recombinant strain DH5α/pF1845 (expressing the fimbrial adhesin F1845) similarly induced time-dependent phosphatidylserine (PS) externalization, suggesting a major specific role of this virulence factor. Using small interfering RNA (siRNA) decay-accelerating factor (DAF)-transfected PLB-985 cells, we then showed that this PS externalization was triggered in part by glycosylphosphatidylinositol (GPI)-anchored DAF receptor engagement (leading to tyrosine kinase and protein kinase C activation) and that it required cytoskeleton and lipid raft architectural integrity. PS externalization under these conditions was not dependent on caspases, mitochondria, lysosomes, or reactive oxygen or nitrogen species. F1845-mediated PS externalization was sufficient to enable macrophage engulfment of infected differentiated PLB-985 cells. These findings provide new insights into the neutrophil response to Afa/Dr DAEC infection and highlight a new role for F1845 fimbriae. Interestingly, although apoptosis pathways were not engaged, C1845-infected PLB-985 cells displayed enhanced removal by macrophages, a process that may participate in the resolution of Afa/Dr DAEC infection and related inflammation.Strains of diffusely adhering Escherichia coli expressing Afa/Dr fimbriae (Afa/Dr DAEC) belong to group 6 of the pathogenic E. coli (35). They can cause childhood diarrhea and are responsible for one-third of recurrent urinary tract infections in adults (53). In vitro, enteric wild-type Afa/Dr DAEC strain C1845, which bears F1845 fimbriae, triggers decay-accelerating factor (DAF)-dependent and mitogen-activated protein kinase (MAPK)-dependent interleukin-8 (IL-8) synthesis by polarized colonic epithelial T84 cell monolayers. This leads to transepithelial migration of human polymorphonuclear neutrophils (PMN), which in turn induce epithelial synthesis of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and IL-1β (4, 5). These interactions between PMN and apical enterovirulent E. coli colonizing the intestinal brush border were documented only recently. First, Brest et al. obtained evidence that Afa/Dr DAEC could modulate PMN apoptosis and was inefficiently engulfed by PMN (8). Then, using the human myeloid cell line PLB-985 differentiated into fully mature PMN, our group found that Afa/Dr DAEC could activate PMN, triggering an oxidative burst and rapid release of preformed myeloperoxidase and IL-8, followed by IL-1α, TNF-α, and IL-8 synthesis (52); type 1 pili were identified as the promoting bacterial virulence factor, and DAF was identified as the PMN membrane-bound receptor that triggers cell signaling via Erk1/2 and p38 MAPKs, Src tyrosine kinase, and NF-κB.Proinflammatory responses might contribute to inducing and perpetuating local gut inflammation. Indeed, delayed death and clearance of infected PMN in tissues can cause exaggerated inflammation and prolonged infection (15); in particular, enzymes and reactive oxygen species (ROS) produced by PMN can damage surrounding tissues. Alternatively, a decrease in the PMN life span due to rapid apoptosis can be a contributing factor in severe and recurrent infections (39). PMN become apoptotic and are then recognized, engulfed, and cleared by professional phagocytes, such as tissue macrophages, which prevents them from releasing their toxic contents (22). The detection, recognition, and ingestion of apoptotic cells involve at least three “eat me” molecules, namely, phosphatidylserine (PS), endocytic receptors, and soluble molecules bridging apoptotic PMN and macrophages (33). A nonapoptotic PS externalization mechanism has also been described, which allows PMN engulfment by macrophages in certain conditions (34, 54, 58). Many microbial pathogens have evolved to circumvent PMN attack through six main strategies: activation of survival and stress responses, contact avoidance, phagocytosis prevention, intracellular survival, PMN death induction, and evasion of PMN extracellular traps (32, 60). Pathogen-induced stimulation of host cell death pathways may eliminate key immune cells or be involved in evasion of other host defenses, while, on the other hand, suppression of death pathways may facilitate the proliferation of intracellular pathogens (20, 36).Here, we report that E. coli wild-type strain C1845 and its recombinant counterpart DH5α/pF1845, which harbors a plasmid encoding F1845 fimbriae, similarly induce time-dependent PS externalization on differentiated PLB-985 cells, suggesting a role for the F1845 adhesin. Further investigation showed that PS externalization followed interaction between F1845 fimbriae and PLB-985 cell membrane-bound DAF. F1845-induced DAF-dependent PS externalization involved tyrosine kinase and protein kinase C (PKC) activation and required cytoskeleton and lipid raft integrity. We also found that PS externalization was not related to three of the main apoptosis pathways (caspase activation and the mitochondrial and lysosomal pathways) or to release of reactive oxygen or nitrogen species. Finally, we showed that the nonapoptotic PS externalization enabled macrophage engulfment of infected PLB-985 cells. Together, these results suggest that this PMN response could participate in resolution of Afa/Dr DAEC infection and the related inflammation.     

Expression of apoptosis‐related genes in an Ethiopian cohort study correlates with tuberculosis clinical status

Mycobacterium tuberculosis remains one of the world's deadliest pathogens in part because of its ability to persist in the face of an active immune response. It has been suggested that apoptosis of infected macrophages is one way in which the host deals with intracellular pathogens and that M. tuberculosis can inhibit this process. To assess the relevance of this process for human disease, we compared the expression of multiple genes involved in the activation of the extrinsic (“death receptor initiated”) pathway of apoptosis in 29 tuberculosis patients, 70 tuberculosis contacts and 27 community controls from Ethiopia. We found that there is a strong upregulation of genes for factors that promote apoptosis in PBMC from individuals with active disease, including TNF‐α and its receptors, Fas and FasL and pro‐Caspase 8. The anti‐apoptotic factor FLIP, however, was also upregulated. A possible explanation for this dichotomy was given by fractionation of PBMC using CD14, which suggests that macrophage/monocytes may regulate several key molecules differently from non‐monocytic cells (especially TNF‐α and its receptors, a finding confirmed by protein ELISA) potentially reducing the sensitivity to apoptotic death of monocyte/macrophages – the primary host cell for M. tuberculosis. This may represent an important survival strategy for the pathogen.     

Intracellular complement − the complosome − in immune cell regulation

The complement system was defined over a century ago based on its ability to “complement” the antibody-mediated and cell-mediated immune responses against pathogens. Today our understanding of this ancient part of innate immunity has changed substantially and we know now that complement plays an undisputed pivotal role in the regulation of both innate and adaptive immunity. The complement system consists of over 50 blood-circulating, cell-surface expressed and intracellular proteins. It is key in the recognition and elimination of invading pathogens, also in the removal of self-derived danger such as apoptotic cells, and it supports innate immune responses and the initiation of the general inflammatory reactions. The long prevailing classic view of complement was that of a serum-operative danger sensor and first line of defence system, however, recent experimental and clinical evidences have demonstrated that “local” tissue and surprisingly intracellular complement (the complosome) activation impacts on normal cell physiology. This review will focus on novel aspects of intracellular complement activation and its unexpected roles in basic cell processes such as metabolism. We also discuss what the existence of the complosome potentially means for how the host handles intracellular pathogens such as viruses.     

Mechanisms of Native and Acquired Resistance to Infection with Cryptococcus Neoformans

Abstract

The mechanisms of resistance mustered by the host to ward off infection with Cryptococcus neoformans mirror many of the classical responses shown with intracellular bacteria. Thus, the administration of an appropriate cellular antigen can protect animals from an otherwise lethal infection in the absence of demonstrable humoral antibody. It is now known that this resistance most probably can be transferred both by “immune” lymphocytes and macrophages.³⁶ A similar effect can be obtained with endotoxin, but limitation of the infection in this case apparently requires the presence of circulating antibody directed against the capsule. Neutralization of the adverse effects of extracellular polysaccharide on chemotaxis and pinocytosis is the most plausible explanation.

In spite of the formidable natural protection afforded by the respiratory apparatus, the ultimate mediators in defense against C. neoformans must be the phagocytes, most particularly the macrophages. Although the metabolic activity of fully activated macrophages has never been the subject of a detailed study, basic proteins isolated from both PMN and mononuclear phagocytes can kill the fungus, both in vitro and apparently also in vivo, in appropriate intra and extracellular situations.     

“Pro-eu-karyote” graft acceptance: A mechanism for intracellular parasitism — A new hypothesis for pathogenesis of leprosy

The capacity to recognize “self” and “non-self”, that is present even in unicellular organisms, plays a key role in biological evolution and maintenance of species. As soon as lymphocytes made their appearance in phylogeny, they became an integral part of the process of rejection of foreign grafts. A number of vertebrates also possess a well-defined system of tissue histocompatibility antigens that determines the degree of foreigness in grafted cells. It is well established that donor cells could only survive if they are accepted as “self” by the recipient. This concept is now extended to interaction of prokaryotes (microbes, protozoa, etc.) with mammalian cells. It is hypothesized that intracellular parasites survive and multiply only in those cells which recognize them (parasites) as “self”. Once a parasite enters the susceptible cell, a situation similar to a cellular graft in unicellular organism is created and the host immune system might be of little help, particularly if the affected cells were cells other than macrophages. The basic mechanism of defense in such a situation may reside in a “self”, “non-self” recognition system. Those who recognize the parasite as “non-self” would be able to kill it. The mechanisms of “self” “non-self” recognition, as well as that of killing of “non-self” prokaryotes, need further elucidation. Clinico-pathological features of leprosy, including the Mitsuda reaction, which is the local response of body to surface (transplantation/recognition) antigens of $\text{M.}$$\text{leprae}$, have many features that support this notion.     

Structural study of TTR-52 reveals the mechanism by which a bridging molecule mediates apoptotic cell engulfment

During apoptosis, apoptotic cells are removed by professional phagocytes or neighboring engulfing cells either directly through phagocytic receptors or indirectly through bridging molecules that cross-link dying cells to phagocytes. However, how bridging molecules recognize "eat me" signals and phagocytic receptors to mediate engulfment remains unclear. Here, we report the structural and functional studies of Caenorhabditis elegans TTR-52, a recently identified bridging molecule that cross-links surface-exposed phosphatidylserine (PtdSer) on apoptotic cells to the CED-1 receptor on phagocytes. Crystal structure studies show that TTR-52 has an open β-barrel-like structure with some similarities to the PKCα-C2 domain. TTR-52 is proposed to bind PtdSer via an "ion-mediating" PtdSer-binding mode. Intensive functional studies show that CED-1 binds TTR-52 through its N-terminal EMI domain and that the hydrophobic region of the TTR-52 C terminus is involved in this interaction. In addition, unlike other PtdSer-binding domains, TTR-52 forms dimers, and its dimerization is important for its function in vivo. Our results reveal the first full-length structure of a bridging molecule and the mechanism underlying bridging molecule-mediated apoptotic cell recognition.     

Dendritic cells in Leishmania major-immune mice harbor persistent parasites and mediate an antigen-specific T cell immune response

Upon infection with Leishmania major, a cause of human cutaneous leishmaniasis, mice of resistant strains are able to control the infection, with lesions resolving spontaneously. A long-lasting cell-mediated immunity protects them from reinfection. Nevertheless, small numbers of viable parasites persist in the lymph nodes of these mice. We have recently documented that, in addition to macrophages, epidermal Langerhans cells can ingest L. major. Furthermore, Langerhans cells have the unique ability to transport viable parasites from the infected skin to the draining lymph node for presentation to antigen-specific T cells and initiation of the cellular immune response. During migration, Langerhans cells develop into dendritic cells. In the present study, we analyzed whether dendritic cells support the persistence of parasites in immune hosts. Immunohistological studies and assays in vitro showed that in the lymph nodes of mice that have recovered from infection with L. major, both macrophages and dendritic cells harbor viable parasites. However, only dendritic cells were able to induce a vigorous T-cell immune response to L. major in vitro in the absence of exogenous antigen. Tracking experiments conducted in vivo suggested that the infected dendritic cells in the lymph nodes are derived from Langerhans cells that have emigrated from the skin. The data demonstrate that L. major-infected dendritic cells and macrophages in lymph nodes of immune animals represent long-term host cells, but only dendritic cells have the ability to present endogenous parasite antigen to T cells. Long-term infected dendritic cells may thus allow the sustained stimulation of a population of parasite-specific T cells, protecting the mice from reinfection. Our results favor the hypothesis that the persistence of antigen supports the maintenance of T cell memory and that dendritic cells are critically involved in this process.     

Intracellular Survival of Leishmania major in Neutrophil Granulocytes after Uptake in the Absence of Heat-Labile Serum Factors

The role of polymorphonuclear neutrophil granulocytes (PMN) in defense against the intracellular parasite Leishmania is poorly understood. In the present study, the interaction of human PMN with Leishmania major promastigotes was investigated in vitro. In the presence of fresh human serum, about 50% of PMN phagocytosed the parasites within 10 min and the parasite uptake led to PMN activation, resulting in the killing of most ingested parasites. Heat inactivation of the serum markedly reduced the rate of early parasite phagocytosis, suggesting a role of complement components in the early uptake of LEISHMANIA: However, over 50% of PMN were able to ingest parasites in the presence of heat-inactivated serum if the coincubation was extended to 3 h. After 3 h, 10% of the PMN were found to internalize Leishmania even under serum-free conditions. These findings indicate that PMN possess mechanisms for both opsonin/complement-dependent and -independent uptake of LEISHMANIA: Both pathways of uptake could be partially blocked by anti-CR3 antibody. Mannan-binding lectin was found not to be involved in this process. When phagocytosed in the absence of opsonin, the majority of Leishmania parasites survived intracellularly in PMN for at least 1 day. These data suggest a dual role of PMN in the early response to L. major infection. On the one hand, PMN can rapidly eliminate the intracellular parasites, and on the other hand, Leishmania can survive intracellularly in PMN. These data, together with the finding that intact parasites were seen in PMN isolated from the skin of infected mice, suggest that PMN can serve as host cells for the intracellular survival of Leishmania within the first hours or days after infection.