Regarding the *trans*-infection cell-to-cell process from DCs, several studies showed that DCs can capture HIV-1 through binding of the viral envelope glycoproteins to the mannose specific C-type lectin receptor (DC-SIGN) and store viruses into tetraspanin-enriched compartments, in continuity with the plasma membrane without viral replication in recipient DCs (58, 83, 84, 87). More recently, it has been reported that the immunoglobulin(I)-type lectin Siglec-1 (or CD169) can also bind to HIV-1 particles carrying sialyl-lactose gangliosides on their envelope (88–90). After capture, viruses remain at the cell surface or are internalized in vesicular containing compartments (see below). Then, DCs are able to transfer these viruses to CD4+ T cells independently of viral replication through the formation of an "infectious" synapse (91–94), thus decreasing the efficiency of broadly neutralizing antibodies on HIV-1 infection of target T cells (95).

While the formation of the virological synapse depends on the interaction between the viral gp120 envelope glycoprotein and the CD4 receptor, the infectious synapse formed during *trans*-infection does not rely upon CD4/gp120 interaction since Rodriguez-Plata et al. showed that the number of cellto-cell conjugates formed between DCs and CD4+ T cells was not increased in the presence of HIV-1 (85). However, they also reported that the formation of the infectious synapse was decreased by 60% when the interaction between the adhesion molecules ICAM-1 and LFA-1 was disrupted. Furthermore, recognition of MHC-superantigen complexes presented at the surface of DCs by the TCR significantly enhanced virus transfer across the infectious synapse (85, 91). These authors suggest that the formation of the infectious synapse is not triggered by the virus but is more related to a hijacking of the immunological synapse by HIV-1. However, their experiments do not prove formally the formation of a canonical immunological synapse in their system as TCR or signaling molecules recruitment at the interface was not verified. Moreover, a previous study failed to detect any MHC-II or TCR recruitment at the interface between DCs carrying viruses and target CD4+ T cells, thus excluding the identification of this structure as a *bone fide* immunological synapse (87).

While the formation of the infectious synapse does not rely on CD4/gp120 interaction, this interaction is still required for efficient productive infection of CD4+ T cells following formation of cell-to-cell conjugates with DCs carrying HIV-1. After the formation of conjugates, the CD4 receptor and CXCR4 or CCR5 co-receptors are recruited at the site of the cell–cell contact (96). Furthermore, intercellular transfer to CD4+ T cells of virus particles captured by DCs can be blocked using high concentrations of broadly neutralizing antibodies targeting the gp120 and gp41 envelope glycoproteins (97).

Rearrangements of the actin cytoskeleton also play a key role in HIV-1 transfer across the infectious synapse. A recent screening shows that tetraspanin 7 and dynamin-2 control nucleation and cortical stabilization of actin to maintain viruses onto dendrites for efficient cell-to-cell transfer from DCs to CD4+ T cells when cells are cocultured, and simultaneously infected with HIV-1 (98). Furthermore, it has been shown that large sheet-like membrane structures derived from DCs carrying HIV-1 wrap around T cells leading to a large interface at the infectious synapse (58). Within this interface, filopodia extensions from T cells are able to interact with the virus-containing compartments (VCCs) in continuity with the plasma membrane of DCs for efficient cell-to-cell transmission (58). Together, these results show that in DCs, HIV-1 can be efficiently transferred by hijacking the immunological synapse without productive infection of DCs.

As specialized DCs present in cervico-vaginal and rectal mucosa, Langerhans cells have been proposed to be the first targets of HIV-1 after sexual exposure. However, these cells were proposed to capture and degrade captured virions through expression of the DC-SIGN-related lectin Langerin at their surface, avoiding virus transmission from Langerhans cells to CD4+ T cells (99). More recently, in an *ex vivo* human tissue explant model, Ballweber et al. showed that vaginal Langerhans cells were able to transmit HIV-1 to CD4+ T cells, without being productively infected, rendering HIV-1 particles insensitive to inhibition by reverse transcriptase inhibitors in this context (100).

Of note, some studies suggest that the expression of Siglec-1 in macrophages mediates the internalization of HIV-1 particles in a VCC. As for DCs-to-T-cell transmission, the presence of these storage compartments in macrophages allows for the transfer of virions to CD4+ T cells (3, 101). Interestingly, the role of Siglec-1 for viral capture by macrophages has been evidenced *in vivo* in lymph node from HIV-1 or MLV infected mice (90). By showing subsequent transfer of MLV from macrophages to B cells, Sewald et al. proposed that this mechanism could be transposed to HIV-1 macrophage-to-T-cell transmission (90).

#### HIV-1 Transfer through Conjugates Formed with Macrophages

Compared with the cell-to-cell transmission mechanisms of HIV-1 involving T cells or DCs as infected donor cells, little is known about the formation of conjugates between HIV-1 infected macrophages and target CD4+ T cells. As evidenced by the presence of infected macrophages in different tissues in infected patients, macrophages are cellular targets of HIV-1 and probably play an important role in HIV-1 pathogenesis (102–108). Like other infected cells, they participate in cell-tocell transmission of HIV-1 and are essential for HIV-1 spread in tissues of infected patients. One major feature of HIV-1-infected macrophages is related to the assembly and budding steps of the new viral particles. While in T cells viral assembly takes place at the plasma membrane, *de novo* formed viral particles accumulate both at the plasma membrane and in tetraspaninenriched compartments called VCCs in productively infected macrophages (109–112). These VCCs, which do not exhibit the same characteristics than endosomes or multivesicular bodies, but are characterized by the presence of the tetraspanins CD63 and CD81 (113–116), and are connected to the extracellular space by thin channels in continuity with the plasma membrane (110, 117, 118). Viruses thus assemble in macrophages in a protected compartment and are then released in the extracellular environment.

Infected macrophages can efficiently transfer viruses to uninfected CD4+ T cells across the formation of a virological synapse but the mechanisms involved in the formation of this macrophage/T-cell virological synapse are not completely characterized (3, 119). During the formation of the conjugates between infected macrophages and target T cells, VCCs could rapidly move to the virological synapse (82) through an actin cytoskeleton-dependent mechanism (81). Similarly to the virological synapse formed between T cells, the virological synapse involving virus-donor macrophages leads to the recruitment of CD4, CCR5, LFA-1, and ICAM-1, as well as the viral Gag precursor and envelope glycoproteins at the site of cell–cell contact (81, 119). In contrast to the virological synapse formed between T cells, which is dependent of CD4, the formation of conjugates between infected macrophages and CD4+ T cells appears to be independent of gp120 (82), while the viral transfer is dependent of gp120/CD4 and LFA-1/ICAM-1 interactions (81). Through the formation of the virological synapse, infected macrophages can transfer a high multiplicity of HIV-1 to CD4+ T cells, promoting reduced viral sensitivity to reverse transcriptase inhibitors as well as to a panel of neutralizing antibodies (81, 82). While the virological synapse between infected macrophage and T-cell targets show several differences with the virological synapse between T cells (82, 119, 120), no similar structure has been described so far, to our knowledge, between infected T cells and uninfected macrophages for virus cell-to-cell transfer in these HIV-1 target cells.

#### ENGULFMENT OF INFECTED T CELLS BY MACROPHAGES AND ENTOSIS

Despite the fact that HIV-1 infection of T cells by cell-to-cell transfer has been largely documented, cell-to-cell infection of macrophages remains poorly investigated. Recently, the group of Quentin Sattentau pointed out for the first time a new mechanism of specific cell-to-cell transfer of HIV-1 from infected CD4+ T cells to macrophage targets (121) (**Figure 1F**). The authors showed that macrophages could engulf HIV-1-infected T cells leading to productive infection of the macrophage targets. The engulfment of infected healthy but rather dying T cells was significantly higher compared with uninfected healthy T cells, indicating that the cell death and infection of virus-donor T cells might independently promotes T-cell engulfment by macrophages. This preferential uptake of infected dying T cells was independent of the interaction between gp120 and the CD4 receptor but was dependent of remodeling of the actin cytoskeleton. Since this engulfment/ uptake of infected T cells by macrophages was dependent of actin remodeling and was not inhibited by amiloride, an inhibitor of macropinocytosis, the authors suggested that this engulfment process likely results from phagocytosis mechanisms.

While the uptake of infected T cell by macrophages was independent of the Env/CD4 interaction, the productive infection of macrophages following uptake of T cells would be dependent of this interaction, and also depended of the tropism of the viruses. The uptake of infected T cells was evidenced by a concentrated localization of the viral Gag precursor in the macrophage target observed in association with the CD3 T-cell specific marker. The engulfment of T cells infected with CCR5-tropic viruses led to productive infection of the macrophage targets able to produce infectious virus particles in the cell-culture supernatant. In contrast, after engulfment of T cells infected with CXCR4-tropic viruses, no productive infection of the macrophage targets was observed (121). From these data, it appears evident that macrophages can be productively infected by phagocytosis of HIV-1-infected T cells, but the mechanisms for virus entry in macrophages from intracellular phagocytosed T cells remains to be defined. Nonetheless, the dependency of co-receptor usage by HIV-1 particles for productive infection could indicate a fusion of viral particles with the phagosome membrane.

Phagocytosis of SIV-infected T cells by myeloid cells *in vivo* was also suggested by the group of Jason Brenchley in SIVinfected macaque models (122, 123). Their studies point out the presence of viral RNA and DNA originating from T cells in myeloid cells from spleen and lymph nodes of infected monkeys, but do not formally prove that this presence of specific markers was related to a phagocytosis mechanism of SIV-infected T cells by myeloid cells, since no *per se* phagocytosis experiment was carried out in these studies.

Of note, another type of engulfment of HIV-1-infected CD4+ T cells by epithelial cells *via* an entosis mechanism has been observed. Entosis refers to the invasion of non-apoptotic cells into another live cell and then induces the formation of cell-in-cell structures (124, 125). Using infected cells from an immortalized T-cell line, Ni et al. reported that epithelial cells could internalize infected T cells *in vitro*, as well as *in vivo* in sigmoid colon explants from HIV-1-infected patients showing the formation of cell-in-cell structures (126). Through this entosis process, the epithelial cells could be productively infected, as evidenced by the diffuse intracytoplasmic staining of the viral capsid protein.

#### CELL–CELL FUSION

### Formation of T-Cell Syncytia

Cell-to-cell fusion between infected CD4+ T cells, or between infected and uninfected CD4+ T cells, has been initially proposed to be another mechanism for HIV-1 infection and dissemination between T cells (127–131). Early studies suggested that infected T cells could fuse with uninfected T cells to form giant syncytia, 5–100 times bigger than individual cells (131, 132). In this context, cell-to-cell fusion occurs through actin cytoskeleton rearrangements, is dependent of LFA-1/ICAM-1 interaction and is mediated through interaction between envelope glycoproteins expressed at the cell surface of infected cells and CD4 expressed on the target cells (127, 129, 131, 133). Yet, these T-cell syncytia, initially observed only *in vitro* with infected immortalized T-cell lines, have been shown to die rapidly by a mitochondriondependent apoptosis mechanism (134, 135). The formation of HIV-1 T cell syncytia leads to activation of the serine-threonine kinase, mTOR, which mediates phosphorylation of the transcription factor p53. Thus, p53 induces upregulation of the proapoptotic regulator Bax leading to mitochondrial permeabilization and release of pro-apoptotic mitochondrial proteins (136, 137). Several groups thus proposed that this cytopathic effect related to the formation of T-cell syncytia could be the mechanism of the CD4+ T cells loss observed during infection in HIV-1-infected patients (127, 128, 132).

However, formation of T-cell syncytia has been a controversial subject since other studies did not observed formation of T-cell syncytia using HIV-1-infected primary CD4+ T cells (3, 4, 11). Because no evidence of the formation of such T-cell syncytia was reported *in vivo*, it has been suggested that these giant syncytia could be *in vitro* artifacts only observed with immortalized cell lines and restricted to CXCR4-viruses (138). Viral strains were indeed initially classified as syncytia-inducing or non-syncytia inducing (NSI) strains, referring to their capacity to induce syncytia *in vitro*, and then to their ability to use either CXCR4 or CCR5 for virus entry, respectively (139). However, cells infected with NSI viruses can readily form syncytia with CCR5-positive cells, and a new classification of viral strains based on CXCR4 or CCR5-usage was adopted (140).

Interestingly, small T-cell syncytia, containing no more than five nuclei, have been then observed *in vivo* in lymph nodes from HIV-1-infected patients (141). In addition, some more recent studies using HIV-1-infected "humanized mouse models" reported the presence of motile infected syncytia in lymph nodes, smaller than those observed *in vitro* (12). These motile small T-cell syncytia can establish tethering interactions with uninfected T cells that may facilitate cell-to-cell transmission through the formation of a virological synapse (12, 142). Interestingly, these interactions between infected small syncytia and uninfected T cells do not lead to cell-to-cell fusion suggesting that this mechanism of cell fusion is finely regulated (12).

### Inhibition of T-Cell Fusion at the Virological Synapse

The numerous studies describing the formation of conjugates between infected and uninfected T cells for viral transfer across the virological synapse do not present observations of cell–cell fusion between the virus-donor T cells expressing the viral envelope glycoproteins and the target T cells expressing the CD4 receptor and CXCR4 and CCR5 co-receptors, confirming that this process is finely regulated during the formation of the virological synapse between T cells.

Since HIV-1 is a highly fusogenic virus that is preferentially transmitted by direct contact between two cells, the mechanisms inhibiting the fusion between an infected cell and a target cell deserves to be questioned. Indeed, in most of the T cell-to-T-cell transmission of HIV-1, a very few syncytia are observed compared with the formation of virological synapses (3, 4, 11). Moreover, large syncytia formed with T cells were characterized as rapid-dying cells (134, 136), which might indicate that HIV-1 has developed some mechanisms to inhibit the formation of these large T-cell syncytia to survive in infected host. As usually, it was proposed that HIV-1 took advantage of the intracellular systems displayed by its host, and used molecular mechanism to inhibit cell–cell fusion.

For example, the CD81 tetraspanin membrane protein is recruited at the site of cell–cell contact during HIV-1 cellto-cell transmission and was proposed to be involved in the regulation of fusion (143). CD81 knockdown or inhibition with a specific antibody dramatically increased the capacity of infected T cells to fuse with surrounding non-infected cells, indicating that CD81 is a negative regulator of T cell–T cell fusion during virological synapse formation. CD81 expression at the virological synapse seems to be regulated by the actin/ plasma membrane connector, ezrin. Accordingly, reduction of ezrin expression in T cells decreased the level of CD81 localized at virological synapses, thus increasing cell–cell fusion between infected T cells (144). Other tetraspanin proteins such as CD9, CD63, or CD82, have been also studied for their role in HIV-1-mediated cell–cell fusion (144, 145). In addition, it has been shown that CD63 may interact with the CXCR4 coreceptor and induces downregulation of CXCR4 expression at the cell surface, resulting in the inhibition of HIV-1 infection (146, 147). Like CD81, these molecules may act as regulators of cell fusion, but most of the experiments have been performed using HeLa or HEK293T non-lymphoid cell lines. Therefore, experiments need to be performed in more relevant cell models to precisely define the molecular requirement of these tetraspanins in the regulation of syncytia formation between HIV-1-infected T cells.

### Presence of Multinucleated Giant Cells (MGCs) in Infected Tissues

The mechanism of T-cell syncytia formation has been largely documented *in vitro* and discussed for its *in vivo* relevance. Nonetheless, processes of cell–cell fusion for HIV-1 infection and dissemination are not restricted to T cells. Some studies showed that infected multinucleated macrophages, as well as multinucleated DCs, could be found in different tissues *in vivo* in HIV-1-infected patients (148, 149). The presence of HIV-1-infected multinucleated syncytia expressing specific DC markers was found at the surface of the nasopharyngeal tonsils of HIV-1-infected patients and reported 20 years ago (148). The same group reported that infected T cells could fuse *in vitro* with skin-derived DCs (94), thus leading to multinucleated cell formation between T cells and DCs. Similarly, multinucleated giant HIV-1-infected macrophages have been found *in vivo* during infection in several different tissues, including lymph nodes, spleen, lungs, genital, and digestive tracts, and the central nervous system (CNS) (102–106, 108, 148, 150–153). While several groups showed the presence of infected multinucleated macrophages in tissues, and more specifically in the brain of HIV-1-infected patients and SIVinfected monkeys, the cellular and molecular mechanisms related to their formation remained poorly investigated, with only one *in vitro* study demonstrating a role for the HIV-1 auxiliary protein Nef in the formation of multinucleated macrophages (154).

We recently reported *in vitro* original results and proposed a model for the formation of HIV-1-infected MGCs related to a two-step cell–cell fusion process for HIV-1 cell-to-cell transfer from infected T cells and viral dissemination in macrophage targets (155) (**Figure 1G**). First, a heterotypic fusion occurs between HIV-1-infected T cells and macrophage targets for virus transfer. Then the newly formed lymphocyte/macrophage infected fused cells acquire the ability to fuse with surrounding uninfected macrophages for virus dissemination. As evidenced by cell imaging analyses, the first step is related to the establishment of contacts with infected T cells leading to the fusion of the infected T cells with macrophages. This cell fusion, dependent of the viral envelope/CD4 receptor interaction and restricted to CCR5-tropic viral strains, has been evidenced by a massive and fast transfer of Gag + material, as well as the presence of specific T cells markers (CD3, CD2, and Lck) in the cytoplasm and at the plasma membrane of the macrophage targets. The newly formed lymphocyte/macrophage fused cells (LMFCs) are then able to fuse with surrounding uninfected macrophages. These two sequential cell fusion processes are dependent on the viral envelope and lead to the formation of highly virusproductive MGCs that could survive for a long time in host tissues to produce infectious virus particles as shown *in vivo* in lymphoid organs and the CNS of HIV-1-infected patients and SIV-infected macaques (103, 106, 108, 152, 156). Similarly, the first step related to the initial T cell-to-macrophage fusion agrees with results showing that myeloid cells from lymphoid tissues of SIV-infected macaques contain T-cell markers and viral RNA and DNA originating from infected T cells (122, 123). This route of infection may be a major determinant *in vivo* for virus dissemination and establishment of macrophage virus reservoirs in host tissues.

#### CONCLUSION

Since the HIV-1 discovery, a lot of works have been accomplished to decipher its life cycle, in order to counteract virus transmission and dissemination. Counteraction of host defenses is a major feature of HIV-1, since it is able to infect a large panel of immune cells and to largely decrease their effector functions. During the past years, several entry pathways for virus dissemination have been discovered but still need further investigations. While HIV-1 was initially suspected to enter its target cells only through fusion of viral particles with host T-cell membranes, it appears more and more evident that it can use a large diversity of cellular features to infect target cells, especially through direct cell-to-cell transmission by close contacts between an infected virus-donor cell and a target cell. Cell-to-cell transfer of HIV-1 indeed allows a massive release of viral material toward the target cell inducing a strong increase of viral infectivity, compared with infection with cell-free viruses. Depending on the infected donor cells as well as the target cell lineages, HIV-1 can take advantage of their specific physiology and features to infect its host. In T cell-to-T-cell transmission, HIV-1 provokes the formation of contacts, through Env–CD4 interactions, to allow a massive transfer of viruses between the two cells, thus improving its ability to enter the host cell. This intercellular structure, called virological synapse, is strongly similar to another but physiological one, the immunological synapse, indicating the HIV-1 ability to hijack T-cell pathways to spread. During trans-infection from DCs to target T cells, HIV-1 is even able to re-use at its advantage the interaction between APCs and T cells to cross the intercellular space for infection of target T cells. Through the interface formed between donor and target cells, filopodia coming from target T cells penetrate some VCCs in donor DCs to capture virions.

Therefore, intercellular communication pathways seem to be a road of choice for HIV-1 spreading. By increasing the frequency of filopodia formation in host infected cells, HIV-1 also enhances the surface of contacts of infected cells with the environment, thus improving its efficiency to be transferred to a non-infected neighboring target cell. In addition, HIV-1 can also create real bridges between cells, the so-called TNTs, which resemble to highways for virus transmission and dissemination. Moreover, when infected T cells are engulfed by specializedphagocytic cells such macrophages, instead of being destroyed, HIV-1 is still able to infect the phagocyte.

Finally, HIV-1 uses its own ability to fuse with host cell membranes to provoke fusion between an infected donor cell and a target cell. By this way, HIV-1 is totally hidden form extracellular milieu since the sharing of the cytosol between the two cells appears to be sufficient for viral material dissemination and productive infection the new-formed fused cell able to produce new fully infectious virus particles. While fusion between T cells has been observed since a long time but was controverted as an *in vitro* artifact, more recent studies have pointed out the presence of small T-cell syncytia *in vivo* in lymph nodes from infected patients and proposed that these syncytia are fully able to transmit viral particles by cell-to-cell transfer to other cell targets. Multinucleated macrophages or DCs have also been observed *in vivo* in different tissues from HIV-1-infected patients, indicating the cell-to-cell fusogenicity of HIV-1 for different cell types. We have recently discovered a new mechanism of cell fusion between HIV-1-infected T cells and target macrophages. Heterotypic fusion between these cells appears, at least *in vitro*, to be a massive and very fast process allowing the formation of LMFCs that acquire the ability to fuse rapidly with neighboring non-infected macrophages, thus forming MGCs. The new-formed MGCs can survive for a long period *in vitro* and are highly productive of fully infectious viral particles. This important process could explain the presence of MGCs observed in several tissues of infected patients. Because of their long-time survival capacity, infected MGCs could participate in virus dissemination and establishment of persistent virus reservoirs in host tissues.

To conclude, while cell-associated HIV-1 transmission is believed to contribute to sexual transmission or viral spreading as well as to the formation of latent virus reservoirs, only few studies address the importance of cell-to-cell transfer of HIV-1 *in vivo*, and further investigations are needed to decipher the role of all the intercellular structures and processes described *in vitro* for pathogenesis in HIV-1-infected patients.

### AUTHOR CONTRIBUTIONS

All authors listed, have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.

#### ACKNOWLEDGMENTS

Financial support is provided by INSERM, the CNRS, and the University Paris-Descartes. The team is also funded by Grants from the *Agence Nationale de Recherche sur le SIDA et les Hépatites virales* (ANRS) and Sidaction. LB is supported by grants from the Institut Pasteur International Network and the Chinese Academy of Sciences. MX is supported by a grant from the China Scholarship Council. JB is supported by a grant from Sidaction. The team is part of the International Associated Laboratory (LIA) VIRHOST between CNRS, Institut Pasteur, Université Paris-Descartes, and the Institut Pasteur Shanghai— Chinese Academy of Sciences.

### FUNDING

Financial support is provided by INSERM, the CNRS, and the University Paris-Descartes. The team is also funded by Grants from the Agence Nationale de Recherche sur le SIDA et les Hépatites virales (ANRS) and Sidaction. LB is supported by grants from the Institut Pasteur International Network and the Chinese Academy of Sciences. MX is supported by a grant from the China Scholarship Council. JB is supported by a grant from Sidaction. The team is part of the International Associated Laboratory (LIA) VIRHOST between CNRS, Institut Pasteur, Université Paris-Descartes, and the Institut Pasteur Shanghai—Chinese Academy of Sciences.

#### REFERENCES

cell-to-cell spread at the virological synapse. *PLoS Pathog* (2011) 7:e1002226. doi:10.1371/journal.ppat.1002226

macrophages. *PLoS Pathog* (2008) 4:e1000015. doi:10.1371/journal.ppat. 1000015

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2018 Bracq, Xie, Benichou and Bouchet. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Tunneling nanotubes: intimate Communication between Myeloid Cells

*Maeva Dupont 1,2†, Shanti Souriant1,2†, Geanncarlo Lugo-Villarino1,2, Isabelle Maridonneau-Parini1,2\* and Christel Vérollet1,2\**

*<sup>1</sup> Institut de Pharmacologie et de Biologie Structurale, Université de Toulouse, CNRS, Université Toulouse III Paul Sabatier, Toulouse, France, 2Research Program "IM-TB/HIV" (1167), International Associated Laboratory (LIA), CNRS, Toulouse, France*

#### *Edited by:*

*Christoph Hölscher, Forschungszentrum Borstel (LG), Germany*

#### *Reviewed by:*

*Christian Bogdan, University of Erlangen-Nuremberg, Germany Mario M. D'Elios, University of Florence, Italy*

#### *\*Correspondence:*

*Isabelle Maridonneau-Parini<EMAIL_ADDRESS>Christel Vérollet<EMAIL_ADDRESS>
*† These authors have contributed equally to this work.*

#### *Specialty section:*

*This article was submitted to Microbial Immunology, a section of the journal Frontiers in Immunology*

*Received: 17 October 2017 Accepted: 08 January 2018 Published: 25 January 2018*

#### *Citation:*

*Dupont M, Souriant S, Lugo-Villarino G, Maridonneau-Parini I and Vérollet C (2018) Tunneling Nanotubes: Intimate Communication between Myeloid Cells. Front. Immunol. 9:43. doi: 10.3389/fimmu.2018.00043*

Tunneling nanotubes (TNT) are dynamic connections between cells, which represent a novel route for cell-to-cell communication. A growing body of evidence points TNT towards a role for intercellular exchanges of signals, molecules, organelles, and pathogens, involving them in a diverse array of functions. TNT form among several cell types, including neuronal cells, epithelial cells, and almost all immune cells. In myeloid cells (e.g., macrophages, dendritic cells, and osteoclasts), intercellular communication *via* TNT contributes to their differentiation and immune functions. Importantly, TNT enable myeloid cells to communicate with a targeted neighboring or distant cell, as well as with other cell types, therefore creating a complex variety of cellular exchanges. TNT also contribute to pathogen spread as they serve as "corridors" from a cell to another. Herein, we addressed the complexity of the definition and *in vitro* characterization of TNT in innate immune cells, the different processes involved in their formation, and their relevance *in vivo*. We also assess our current understanding of how TNT participate in immune surveillance and the spread of pathogens, with a particular interest for HIV-1. Overall, despite recent progress in this growing research field, we highlight that further investigation is needed to better unveil the role of TNT in both physiological and pathological conditions.

Keywords: tunneling nanotubes, myeloid cells, innate immunity, pathogens, HIV-1

### INTRODUCTION

Tunneling nanotubes (TNT) represent a novel type of intercellular communication machinery, which differs from the secretion of signaling molecules and the signal transmission through gap or synaptic junctions between adjacent cells. Along with exosomes, TNT mediate long-range communication, independent of soluble factors. They are membranous structures displaying a remarkable capacity to communicate with selected neighbor or distant cells. There are recent reviews covering the broad biological role of TNT, which are able to form in multiple cell types (1–3). Here, our focus is exclusively on TNT formed by myeloid cells, including macrophages, osteoclasts, and dendritic cells (DC). Based on the nascent literature on TNT in these cells, we will discuss the definition of TNT, their mechanisms of formation, and their role in physiological and pathological contexts. We will also address the need of further investigation of these structures to better understand their functions and improve their potential as therapeutic targets in pathological conditions.

#### DEFINITION AND FUNCTION OF TNT

The main obstacle in reviewing the emerging TNT field is the different names given to these structures: TNT, cellular and membrane nanotubes, filopodia bridges, conduits or tubes, and nanotubules. Also, the huge number of publications on carbon nanotubes impedes the track of developments on TNT. Unifying terminology for nanotubes would thus be beneficial. In this minireview, the term TNT will be used as done previously (2, 4). TNT are membranous channels connecting two or more cells over short to long distances. Actually, these structures can extend up to 200 µm in length in macrophages (5). To define TNT, we adopted the three phenotypic criteria proposed in a recent elegant review: (i) they connect at least two cells, (ii) they contain F-actin, and (iii) they do not touch the substrate (2). This definition allows the discrimination of TNT with any other F-actin-rich structures, such as filopodia. Regarding their functional properties, TNT transfer cytoplasmic molecules from one cell to another such as calcium, proteins or miRNA, mitochondria, several vesicles (e.g., lysosomes), pathogens, and cell-surface molecules; this ability constitutes the main functional criterion for TNT definition (6). The end of the structure can form a junctional border with the targeted cell (close-ended TNT) or the cytoplasm of the two connected cells can be mixed (open-ended TNT). On the one hand, the transfer of large molecules such as the lipophilic dye DiO is used to identify open-ended TNT (7). On the other hand, closeended TNT form a junction at their end which are visualized by scanning electron microscopy (8). To avoid the past arguments on the need of cytoplasmic interactions for TNT, we shall consider in this review both close-ended and open-ended TNT (**Figure 1A**). As close-ended TNT mediate signal transfer through distant gap junctions (8, 9), they meet the functional criterion to be considered as TNT. Also, close-ended TNT could represent an intermediary status in the process of open-ended TNT formation. Finally, the group of Davis demonstrated that one particularity of macrophages is their ability to form different classes of TNT: thin ones (<0.7 μm in diameter), containing only F-actin; and thick ones (>0.7 μm), containing F-actin and microtubules (7). These two types of TNT could have different functions, as large material (e.g., lysosomes, mitochondria) can only travel between macrophages *via* thick TNT on microtubules (7).

#### DISCOVERY OF TNT

The first description of functional TNT *in vitro* was made in rat kidney cells (PC12 cells) and human cell lines (10), followed immediately by the identification of similar structures in human monocytes and macrophages (11). It is now clear that TNT can form in several cell types, including cancer cells and most leukocytes. However, to our knowledge, TNT were not described in granulocytes. In DC, TNT appeared to be similar to those made by monocytes-derived macrophages (6, 12). However, unlike DC exposed to anti-inflammatory conditions, only those activated by pro-inflammatory conditions form complex network of TNT able to transfer soluble molecules and pathogens (13). Likewise, macrophages undergo different activation programs within the broad spectrum of pro- (M1) and anti-inflammatory (M2) polarization. Yet, their activation state has not been linked to the formation of TNT. The only available data concern the early HIV-1 infection of macrophages, driving them toward M1 polarization (14) and inducing a significant increase in TNT formation (5, 15–18).

While the majority of studies in TNT biology has been performed in one cell type (homotypic TNT) at a time, TNT formation between different cell types (heterotypic TNT) is not rare. In fact, TNT frequently form between macrophages or DC with another cell type, enabling the exchange of lysosomes, mitochondria, or viral proteins (16, 19–21).

The reason why TNT were discovered quite recently could be attributed to their fragility. Indeed, they are poorly resistant to the existing shearing forces in culture media, as well as light exposure and classical fixation methods. Thus, an appropriate way of performing live imaging is necessary to study TNT. When working on fixed cells, gentle fixation (e.g., glutaraldehyde-based fixation) should help preserve these highly delicate structures (22, 23).

#### FORMATION OF TNT

#### Mechanisms of Formation

Cell examination by time-lapse microscopy suggested two mechanisms of TNT formation could exist. The first one proposes that two cells initially in contact separate from each other, remaining connected through a thin thread of membrane, which will be elongated upon cell separation (**Figure 1A**, right). The second puts forward that a cell would first bulge filopodia and extend them until reaching a neighboring cell, then converting towards TNT after making contact (24, 25) (**Figure 1A**, left). While the former is the prevailing mechanism in lymphoid cells, the latter one is observed in DC as TNT were reported to develop mainly from conversion of their filopodia (13, 19). In the case of macrophages, while they can use both mechanisms (6), the murine macrophage cell line (RAW 264.7 cells) mainly forms TNT from actin-driven protrusions, also called TNT-precursors (26). Of note, these two processes are not necessarily exclusive and could both occur between a given pair of cells. In either case, the requirement of F-actin is not questioned since treatment with latrunculin or cytochalasin D is often used to abolish TNT formation (2, 27, 28).

Regarding the opening of the conduit, and the potential transition between close-ended and open-ended TNT (**Figure 1A**), there is no proposed mechanism available. It is likely that the formation of open-ended TNT involves a step similar to what occurs during virus-to-cell membrane fusion or cell-to-cell fusion (29, 30), eventually leading to the generation of multinucleated giant cells (MGC) (**Figure 1A**).

#### Molecular Actors

Few data are available to describe TNT at the molecular level. M-Sec, also known as tumor necrosis factor-α-induced protein, is one of the best characterized protein involved in TNT formation

close-ended and open-ended TNT formation is still not understood. In addition, TNT could either disconnect cells and thus abrogate their communication or could lead to MGC. (B) Confocal image of day 13 HIV-1-infected human monocyte-derived macrophages and MGC interconnected through a TNT. Arrowheads show a TNT. HIV-1 Gag (red), F-actin (green), DAPI (blue). Scale bar, 50 µm.

in macrophages. Its depletion in Raw264.7 cells reduces the formation of *de novo* TNT and their associated function (transfer of calcium flux) (22). Using the same macrophage cell line, the group of D. Cox recently showed that actin polymerization factors including the Rho GTPases family Rac1 and Cdc42, and their downstream effectors WAVE and WASP, participate in TNT formation (26). In addition, functional TNT are induced by the expression of the leukocyte specific transcript 1 (LST1) protein in HeLa and HEK cell lines. LST1 recruits the actin cross-linking protein filamin and the small GTPase RalA to the plasma membrane where it promotes RalA interaction with the exocyst complex, M-Sec, and myosin; these interactions trigger TNT formation (22, 23). Whether the mechanisms that operate in cell lines derived often from tumor origin apply to primary cells remains to be confirmed.

### *IN VIVO* RELEVANCE OF TNT

A remaining question is to determine to what extent the *in vitro* data available in the literature are relevant *in vivo*. One of the problems is to apply *in vivo* the criteria of *bona fide* TNT (see above), in particular the requirement not to touch the substrate, which seems unlikely in 3D environments*.* In addition, testing the functionality of TNT in the context of tissues is challenging. Therefore, the structures observed *in vivo* should be carefully indicated as "TNT-like structures." Key evidence for TNT-like structures *in vivo* comes from the immunology field providing the first images of thick TNT connecting DC in inflamed mouse corneas (31). To our knowledge, macrophage TNT have not been observed *in vivo* yet. The identification of specific molecular markers for TNT would be a great tool to confirm the existence of these structures *in vivo*. M-Sec, which is involved in TNT formation, cannot be considered as a specific marker since this ubiquitous protein is expressed all over the cytoplasm (5, 18, 28, 32, 33). Thus, one of the priority to progress in the TNT field is to characterize markers allowing unambiguous identification of cell-to-cell tubular connections as TNT.

#### ROLE OF TNT IN PHYSIOLOGICAL CONTEXTS

One of the most studied functions of TNT is the propagation of calcium flux. Calcium signaling through TNT helps regulate cell metabolism and communication between neurons (34). Interestingly, DC present the ability to establish calcium fluxes *via* TNT transmitted within seconds to other DC as far as 500 µm away from the donor cell (12). When TNT are disturbed by M-Sec knockdown, this calcium flux is inhibited (12, 22). DC have also the particularity to form TNT networks allowing the intercellular exchange of antigens (13), including in the context of MHC molecules as described between Hela cells (19, 27). Therefore, TNT could contribute to a higher efficiency in the antigen presentation process to activate adaptive immunity (19).

Another physiological role for TNT concerns the differentiation of osteoclasts (5, 18, 28, 32, 33). Osteoclasts are MGC derived from a myeloid precursor that present the unique ability to degrade the bone matrix, and thus to regulate bone homeostasis. Inhibition of TNT either by latrunculin B or by M-Sec depletion significantly suppresses osteoclastogenesis, and the M-Sec expression level increases during osteoclastogenesis (28, 35). Dendritic cell-specific transmembrane protein, a receptor involved in cell-to-cell fusion, has been shown to be transferred *via* TNT. The authors proposed that this process could participate in cell fusion among osteoclast precursors (28, 35). Moreover, nuclei are found inside large TNT-like structures (36), inferring that they participate in cell-cell fusion to generate OC. Elucidating the role of TNT in differentiation of MGC such as placental trophoblast, myotubes, and osteoclasts could be a new research area.

#### ROLE OF TNT IN PATHOLOGICAL CONTEXTS

Tunneling nanotubes not only contribute to cell-to-cell communication in physiological conditions but also in pathological processes. For example, the transfer of lysosomes from macrophages to fibroblasts, and of mitochondria from mesenchymal stromal cells to macrophages, are mediated by TNT and have important consequences in cystinosis and acute respiratory distress syndrome, respectively (20, 21).

Without the shadow of doubt, the most studied consequence of TNT in diseases is the transfer of pathogens, including prions, bacteria, and viruses [for review, see Ref. (1)]. One of the wellknown example concerns the role of TNT in neurological diseases, especially when caused by prions (34). Actually, in addition to the TNT-dependent transfer of the infectious form of the prion protein (PrPSc) between neuronal cells, TNT support PrPSc transfer from DC to the neurons in which PrPSc is further synthetized and transferred to the rest of the central nervous system (37). Regarding bacteria and viruses, some publications propose that they "surf " along TNT to spread from one cell to another (7, 13, 38–41). For example, in macrophages, live experiments show that *Mycobacterium bovis* bacillus Calmette–Guerin can travel along the surface of thin TNT, toward another macrophage, which will ingest it (7).

Viruses, including HIV-1, are well known to hijack the cytoskeleton in order to enter and travel inside their host cell, as well as towards bystander neighbor cells (5, 33, 39, 41, 42). For example, HIV-1 can actively induce the generation of filopodia in DC to propel virus particles towards neighboring cells. As one of the mechanism of TNT formation starts with membrane extension, filopodia formed upon HIV-1 infection could lead to TNT formation (2), especially in DC that develop networks of TNT from elongation of their dendrites (13, 19). Importantly, the formation of TNT by DC favors trans-infection of targeted CD4<sup>+</sup> T lymphocytes at a relatively long distance, similar to what happens between two distant CD4<sup>+</sup> T lymphocytes (8).

In macrophages, HIV-1 induces TNT formation and potentially uses them to spread (18). Whether thin or thick TNT are formed is unknown. Assuming that thick TNT are induced, HIV-1 could travel inside these structures by using a microtubule-dependent movement, in addition to the described "surfing" of HIV-1 at the surface of TNT. Despite the fact that Gag and Nef proteins and HIV-1-containing vesicles have been detected inside TNT, there are no convincing experiments in living cells available to prove that HIV-1 travels inside TNT and infects the targeted cell (5, 15, 17, 18). Pushing live imaging to super-resolution microscopy techniques would be of great help to study how HIV-1 traffics using TNT.

In light of the importance of macrophages in HIV-1 pathogenesis (43–45), it is crucial to bridge the several gaps that blur our understanding of the role of TNT in macrophages during HIV-1 infection. First, it is important to determine whether HIV-1 induced TNT in macrophages are close- or open-ended to better understand how HIV-1 traffics *via* TNT. Second, whether TNT from a HIV-infected cell could target non-infected cells remains to be elucidated. It would be an efficient way for the virus to spread around without being detected. Finally, the molecular regulation of HIV-1-induced TNT in macrophages has only started to be elucidated. The HIV-1 Nef protein could play a central role in TNT formation by interacting with members of the exocyst complex (16, 18, 46, 47). Moreover, Nef modulates F-actin and cell migration (48), two effects which could participate in TNT generation. Finally, a hallmark of HIV-1 infection is the formation of MGC, a process that can be driven by TNT in order to persist during late infection stages, when most infected macrophages are MGC (**Figure 1B**) (32, 33). Interestingly, both HIV-1-induced TNT and MGC are reduced when macrophages are infected with *nef*-deleted viruses (18, 32, 33).

Importantly, while TNT spread the virus among HIV-1 target cells (T lymphocytes, macrophages, and DC), TNT also affects the nature of infection by circumventing the need for classical receptor-mediated virus entry or transfer viral components to cells that are not susceptible to infection. As a matter of fact, the transfer of Nef *via* TNT between infected macrophages and B cells induces drastic B cell abnormalities at the systemic and mucosal level (16).

#### CONCLUSION

The TNT field requires the unification of the terminology and definition of TNT, as well as the development of new tools adapted for the detection and monitoring of these particular structures. The main challenge so far is to discover molecular markers to specifically identify TNT, especially *in vivo*. To this end, an automated siRNA-based screen could be used in *in vitro* conditions for which TNT formation is controlled, as performed for the virological synapse (49). Another issue is the fragility of TNT which complicates their manipulation. Thus, the use of specific experimental conditions or devices, such as microfluidic systems (50), is needed. Moreover, it would be helpful to study the opening of close-ended TNT in terms of molecular components and dynamics. Likewise, it is imperative to determine whether TNT formation and regulation can be influenced by extracellular stimulti and/or tissue microenvironment in pertinent *in vivo* physiological and pathological contexts. For example, during HIV-1 infection, TNT represent a new way for viral spread.

#### REFERENCES

However, the literature remains scarce, rising far more questions than answers. Interestingly, HIV-1 and other microbes can serve as efficient tools to better understand TNT structure and function. Furthermore, TNT-based studies in the HIV-1 field are needed to better understand viral dissemination and pathogenesis. The particularity of TNT to perform "intimate" communication with a specific partner is probably key in HIV-1 spread. A tempting hypothesis is that infected cells could direct their TNT towards uninfected cells. This way, the virus could spread without being detected by the surveilling immune system. Finally, new insights into the mechanisms of TNT formation and regulation would be of high relevance to design novel therapeutics for several diseases, including viral infections.

#### AUTHOR CONTRIBUTIONS

MD, SS, IM-P, and CV wrote the manuscript. SS created the figure. GL-V edited the manuscript.

#### FUNDING

We are grateful to the TRI imaging facility, in particular Renaud Poincloux and Stéphanie Dauvillier. We thank A. Labrousse, C. A. Spinner, N. Roullet, and B. Raynaud-Messina for critical reading of the manuscript and helpful comments. This work was supported by the Centre National de la Recherche Scientifique, the Agence Nationale de la Recherche (ANR 2010-01301, ANR14-CE11-0020-02, ANR16-CE13-0005-01, ANR-11- EQUIPEX-0003), the Agence Nationale de Recherche sur le Sida et les hépatites virales (ANRS2014-CI-2, ANRS2014-049), the ECOS-Sud program (A14S01), the Fondation pour la Recherche Médicale (DEQ2016 0334894; DEQ2016 0334902), and the Fondation Bettencourt-Schueller. MD is supported by Paul Sabatier University, Toulouse, France, and SS by Sidaction.

presenting a novel route for HIV-1 transmission. *Nat Cell Biol* (2008) 10:211–9. doi:10.1038/ncb1682

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2018 Dupont, Souriant, Lugo-Villarino, Maridonneau-Parini and Vérollet. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

*Javier Martinez-Picado1,2,3†, Paul J. McLaren4,5†, Amalio Telenti6 and Nuria Izquierdo-Useros1 \**

*<sup>1</sup> IrsiCaixa AIDS Research Institute, Badalona, Spain, 2 Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain, 3University of Vic-Central University of Catalonia (UVic-UCC), Vic, Spain, 4National HIV and Retrovirology Laboratory, Public Health Agency of Canada, Winnipeg, MB, Canada, 5Department of Medical Microbiology and Infectious Diseases, University of Manitoba, Winnipeg, MB, Canada, 6Genomic Medicine, J. Craig Venter Institute, La Jolla, CA, United States*

#### *Edited by:*

*Christel Vérollet, UMR5089 Institut de Pharmacologie et de Biologie Structurale (IPBS), France*

#### *Reviewed by:*

*Paul Spearman, Cincinnati Children's Hospital Medical Center, United States Masaaki Miyazawa, Kindai University, Japan*

*\*Correspondence:*

*Nuria Izquierdo-Useros<EMAIL_ADDRESS>
*† These authors have contributed equally to this work.*

#### *Specialty section:*

*This article was submitted to Microbial Immunology, a section of the journal Frontiers in Immunology*

*Received: 14 August 2017 Accepted: 06 November 2017 Published: 21 November 2017*

#### *Citation:*

*Martinez-Picado J, McLaren PJ, Telenti A and Izquierdo-Useros N (2017) Retroviruses As Myeloid Cell Riders: What Natural Human Siglec-1 "Knockouts" Tell Us About Pathogenesis. Front. Immunol. 8:1593. doi: 10.3389/fimmu.2017.01593*

Myeloid cells initiate immune responses and are crucial to control infections. In the case of retroviruses, however, myeloid cells also promote pathogenesis by enabling viral dissemination; a process extensively studied *in vitro* using human immunodeficiency virus type 1 (HIV-1). This viral hijacking mechanism does not rely on productive myeloid cell infection but requires HIV-1 capture *via* Siglec-1/CD169, a receptor expressed on myeloid cells that facilitates the infection of bystander target cells. Murine retroviruses are also recognized by Siglec-1, and this interaction is required for robust retroviral infection *in vivo*. Yet, the relative contribution of Siglec-1-mediated viral dissemination to HIV-1 disease progression remains unclear. The identification of human null individuals lacking working copies of a particular gene enables studying how this loss affects disease progression. Moreover, it can reveal novel antiviral targets whose blockade might be therapeutically effective and safe, since finding null individuals *in natura* uncovers dispensable functions. We previously described a loss-of-function variant in *SIGLEC-*1. Analysis of a large cohort of HIV-1-infected individuals identified homozygous and heterozygous subjects, whose cells were functionally null or partially defective for Siglec-1 activity in HIV-1 capture and transmission *ex vivo*. Nonetheless, analysis of the effect of Siglec-1 truncation on progression to AIDS was not conclusive due to the limited cohort size, the lack of complete clinical records, and the restriction to study only off-therapy periods. Here, we review how the study of loss-of-function variants might serve to illuminate the role of myeloid cells in viral pathogenesis *in vivo* and the challenges ahead.

Keywords: antigen-presenting cell, human immunodeficiency virus type 1, Siglec-1, knockout, genome, human

Antigen-presenting cells (APCs) of the myeloid lineage trigger innate and adaptive immune responses against invading viruses, thus modulating the outcome, progression, and clearance of infections (1, 2). Yet, chronic viral infections counteract several defenses orchestrated by APCs and exploit immunity to favor persistence. Infection caused by the human immunodeficiency virus type

**Abbreviations:** APCs, antigen-presenting cells; HIV-1, human immunodeficiency virus type 1; MLV, murine leukemia virus.

I (HIV-1) is one of the best-studied examples to illustrate this paradox, where APCs act as a double-edged sword throughout the course of infection. Myeloid APCs (such as dendritic cells, monocytes, and macrophages) are not as susceptible to HIV-1 infection as activated CD4<sup>+</sup> T cells (3). This is likely due to host restriction factors such as SAMHD1 (4, 5) that restrict viral infection and decreases myeloid cell capacity for immune sensing (6), limiting the onset of antiviral responses. However, HIV-1 can exploit myeloid APC biology to reach and infect new target cells through a mechanism that does not rely on the productive infection of myeloid cells. This process was described *in vitro* at the early nineties by the laboratory of Dr. Ralph Steinman (7), who received the Nobel Prize for discovering dendritic cells. Despite decades of research though, there is no convincing *in vivo* evidence that demonstrates whether myeloid APCs play a critical role in HIV-1 disease progression.

Upon cellular activation, myeloid APCs can capture and store large numbers of HIV-1 particles (8–10), which are then efficiently transferred to bystander CD4<sup>+</sup> T cells (7, 11, 12) *via* cell-to-cell interactions established as part of their immune surveillance routine. Throughout this process of retention and release of virus, HIV-1 exploits a mechanism by which APCs acquire antigens transported by extracellular secreted microvesicles termed exosomes (13). The acquisition of exosomes by activated myeloid APCs contributes to antigen presentation to CD4<sup>+</sup> T cells (14). This step helps to amplify adaptive immunity without the need for myeloid APCs to be in direct contact with the pathogen (15, 16). Retention of exosomes within intracellular compartments might serve as an antigen depot to control and sustain adaptive immune responses. However, in the case of HIV-1, this internalization route retains infectious particles within protected dynamic compartments (17–19) from where viruses are efficiently transmitted across infectious synapses to susceptible lymphocytes (7, 11, 12).

This particular mode of HIV-1 transmission is known as *trans*-infection; a route that favors *de novo* infection of target cells under circumstances where the same dose of cell-free-viruses do not establish productive infection (11). *Trans*-infection is largely dependent on the expression of the sialic-acid binding I-type lectin receptor Siglec-1 (CD169 or Sialoadhesin) (20–22). Siglec-1 is an interferon inducible receptor constitutively expressed on myeloid cells (23), that is highly upregulated upon myeloid APC exposure to antiviral type I interferons (24, 25). Siglec-1 is a trans-membrane receptor with a long neck that protrudes beyond the glycocalyx of the cell and a terminal V-set domain with the ability to interact with sialylated ligands. While the affinity of Siglec-1 for sialic acid-containing molecules is low, avidity for clusters of sialylated molecules is high (23), allowing for the specific recognition of packaged gangliosides that expose sialyllactose moieties on the viral membrane (26, 27). Likewise, Siglec-1 captures exosomes *via* recognition of sialylated gangliosides packaged on the microvesicle membrane (21), which assemble and bud from cellular membranes. Murine studies have also confirmed the capacity of Siglec-1 expressed on lymphoid tissues to capture exosomes *in vivo* (28). Pioneering reports suggested that DC-SIGN, a C-type lectin expressed on immature DCs that patrol peripheral mucosae in search of invading pathogens, could capture HIV-1 early after viral invasion, travel to lymphoid tissues, and establish productive CD4<sup>+</sup> T cell infection *via trans*-infection (11). While C-type lectins such as DC-SIGN recognize the viral envelope glycoprotein (11), capture of HIV-1 *via* Siglec-1 is independent of this interaction (10, 21). Siglec-1 viral uptake largely exceeds the capacity of C-type lectin receptors for HIV-1 capture (21), making this process much more infectious and underscoring novel scenarios within secondary lymphoid tissues in which Siglec-1 *trans*-infection could fuel viral dissemination.

The molecular pathways governing HIV-1 *trans*-infection *via* Siglec-1 on APCs have been described *in vitro* using both monocyte-derived APCs (20–22) and primary myeloid cells directly isolated from human tissues (25) (**Figure 1**). Another retrovirus, the murine leukemia virus (MLV), also contains sialylated gangliosides and is captured *via* Siglec-1 *in vitro* (29). MLV exploits Siglec-1-mediated *trans*-infection of permissive lymphocytes to establish infection within secondary lymphoid tissues in mice (30) (**Figure 1**). However, the *in vivo*

FIGURE 1 | Siglec-1-mediated retroviral *trans*-infection on distinct myeloid antigen-presenting cells (APCs). Human immunodeficiency virus type 1 (HIV-1) capture *via* Siglec-1 and subsequent transfer to target cells has been reported not only in human APCs derived *in vitro* but also in activated primary myeloid cells isolated *ex vivo*. In murine models, Siglec-1 retroviral *trans*-infection has been reported *ex vivo*, and most importantly, due to the extraordinary ability of Siglec-1 positive APCs to capture cell-free viruses from the lymphatic vessels at the edges of the lymphoid tissue and their capacity to transfer that infectivity to permissive lymphocytes, this mechanism has also been observed *in vivo*.

contribution of Siglec-1 to HIV-1 disease progression remains largely unknown.

The lack of available animal models to study HIV-1 infection makes it challenging to investigate the role of Siglec-1 on HIV-1 pathogenesis *in vivo*. Humanized mouse models susceptible to HIV-1 infection are only established in a few laboratories, and how disease progression in these animals correlates with human pathogenesis needs further investigation. Primate models are also restricted to specific facilities and rely on the use of primate retroviruses that may not directly reflect the biology of HIV-1. Under these circumstances, finding naturally occurring human knockouts could be a good alternative to address the role of key receptors such as Siglec-1 under physiological settings of infection. A deletion in the gene that codes for the HIV-1 co-receptor CCR5, which is needed for acquisition of CCR5-tropic HIV-1, is one of the best-known examples of how a genetic variant alters the phenotype of infection (31). These types of variants have been observed for decades and have also been confirmed by large-scale genomic analysis (32). However, these large-scale analyses have not uncovered novel candidates that might influence HIV-1 disease progression because the available sample sizes are not adequate to assess all possible classes of genetic variation, such as rare and low frequency polymorphisms.

The identification of individuals harboring rare, loss-offunction genetic variants provides an opportunity to study gene function *in vivo*. Recently, large catalogs of sequenced human genomes have demonstrated that individuals carrying homozygous loss-of-function variants, or natural human knockouts, can provide insight into genetic causes of disease and holds tremendous potential for identifying drug targets (33). However, given their generally low frequency, such variants have gone largely undetected in large-scale genomic analyses. As an alternative strategy to identify genes involved in HIV-1 progression, we conducted a search for individuals lacking the expression of Siglec-1 receptor to study the natural course of HIV-1 infection in the absence of this particular receptor (34). We focused on two well-established cohorts of HIV-1 infected individuals that had been longitudinally followed for decades and had extensive clinical records. We identified two homozygous and almost a 100 heterozygous subjects for a particular stop codon variant in the *SIGLEC1* gene. This stop-gain allele is found at highest frequency in individuals of European and South Asian ancestry (1.3%) and is rare or absent in African and East Asian populations (0.5%). *Ex vivo* experiments confirmed that cells from these individuals were functionally null or partially defective for Siglec-1 expression and, consequently, lost their activity in HIV-1 capture and transmission *in vitro*. While the lack of Siglec-1 is likely to abrogate *trans*-infection, the classical HIV-1 infection routes, including cell-free virus infection or cell-tocell HIV-1 transmission still operate in the absence of Siglec-1, explaining the observation of Siglec-1 null individuals that are HIV-1 infected. However, despite the lack of impact on susceptibility, HIV-1 dissemination and disease progression in infected individuals with null or diminished Siglec-1 expression could be delayed compared with wild type individuals*.* Nonetheless, we did not observe an effect of Siglec-1 truncation on progression to AIDS.

Several challenges explain the lack of conclusive results in the study of Siglec-1 genetic variants (**Figure 2**). Power simulations indicate that analysis of a rare variant such as the Siglec-1 allele would require more than 10,000 individuals to detect a relative risk of 5 at *P* < 0.05 under a recessive model—an effect that would be similar to the beneficial outcome of B\*57:01 on HIV-1 control (32, 35–37). This sample size far exceeds even the largest genome-wide studies of HIV-1 progression that comprises ~6,000 patients (38), which does not genotype the Siglec-1 stop variant and cannot be used to impute the presence of this rare allele. Given that the proposed effect requires long-term followup off therapy, it is extremely unlikely that a sufficient sample size could be reached to assess the long-term consequences of the Siglec-1 stop variant on HIV-1 disease. Another limitation faced was the lack of seroconversion date for most of the individuals screened; a clinical record that is normally missing in most cohorts of HIV-1 infected individuals. Thus, disease progression was only followed from the date of diagnosis, which may differ between individuals, especially if they are protected by beneficial phenotypes. Moreover, additional clinical data were missing from key individuals, even though we had focused on cohorts with exhaustive follow-up. Indeed, one of the homozygous individuals found had no clinical records for nine years, and information only resumed after antiretroviral treatment initiation, when viral suppression abrogated any potential effect that the Siglec-1 variant might have had on disease progression. Since current clinical guidelines recommend treatment introduction early after HIV-1 diagnosis (39), in the near future this type of analyses will be restricted to retrospective cohorts, which followed old recommendations and started treatment when CD4 counts dropped below a certain threshold, offering a window of opportunity to monitor the natural course of infection. Finally, complexity also arises from the analysis of phenotypes that can be influenced by the infection of several pathogens at the same time. Indeed, one of the homozygous individuals for the rare Siglec-1 allele had a high CD4<sup>+</sup> T cell count that dramatically dropped when tuberculosis was diagnosed. Lack of Siglec-1 could have had a negative impact on the immune control of the mycobacterial infection, masking any putative beneficial effects caused during HIV-1 progression. Previous studies indicate that Siglec-1 expression on myeloid APCs has a role in combating sialylated bacteria (40, 41). Although sialylation of *Mycobacteria* has not been documented to our knowledge, direct interaction between Siglec-1 and *Mycobacteria* might not be required to impact antibacterial immunity. Alternatively, the lack of Siglec-1 on myeloid APCs could compromise antigen capture *via* exosome or microvesicle transfer and affect the control of the bacterial infection (42–45).

Overall, difficulties and questions faced throughout the study of Siglec-1 null individuals infected with HIV-1 illustrate the major challenges of the field of human knockout genetics applied to infectious diseases. We need to address the biological function of knockout genes of interest *in vivo* and the effect of a particular variant on health-related phenotypes (46). Variability in the observed phenotypes arises not only from the effect that other genetic variants might have on the gene of interest, but also from the exposure to particular environmental conditions,

including the co-occurrence of infections. Animal studies could help to dissect the contribution of these factors by creating the same genetic background and similar environmental conditions in pathogen-free facilities, where co-infections could be experimentally controlled. Working with adequate animal models is, however, complex in the case of HIV-1. An interesting alternative to unambiguously test the potential contribution of myeloid APCs *via* Siglec-1 to HIV-1 disease progression could be to develop antiviral therapeutic agents against Siglec-1. The identification of Siglec-1 null individuals demonstrates that this protein is dispensable, and its therapeutic blockade is therefore expected not to cause serious side effects. Future work targeting Siglec-1 could provide conclusive evidence of the real contribution of myeloid APC to HIV-1 pathogenesis *in vivo*. If proven effective, this new family of antiviral agents against HIV-1 could also offer protection against other retroviral infections by mimicking the loss-of-function mutation found in *SIGLEC1*.

#### REFERENCES

#### AUTHOR CONTRIBUTIONS

JM-P, PM, AT, and NI-U designed the work, prepared the figures, reviewed bibliography, and prepared the manuscript. All the authors approved the final version.

#### ACKNOWLEDGMENTS

The authors are grateful to Dr. M. C. Puertas for critical reading of the manuscript. The authors would like to apologize to all those researchers whose work and contributions were not cited due to space limitations.

#### FUNDING

JM-P and NI-U are supported by the Spanish Secretariat of Science and Innovation through Grant SAF2016-80033-R.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Martinez-Picado, McLaren, Telenti and Izquierdo-Useros. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Functional Analysis of Phagocyte Activity in Whole Blood from hIV/ tuberculosis-Infected Individuals Using a Novel Flow Cytometry-Based Assay

*Ankur Gupta-Wright1,2\*† , Dumizulu Tembo3†, Kondwani C. Jambo1,4, Elizabeth Chimbayo1 , Leonard Mvaya1 , Shannon Caldwell3 , David G. Russell3 \* and Henry C. Mwandumba1,4*

*1College of Medicine, Malawi-Liverpool-Wellcome Trust Clinical Research Programme, Blantyre, Malawi, 2Clinical Research Department, London School of Hygiene and Tropical Medicine, London, United Kingdom, 3Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY, United States, 4Department of Clinical Sciences, Liverpool School of Tropical Medicine, Liverpool, United Kingdom*

#### *Edited by:*

*Christel Vérollet, UMR5089 Institut de Pharmacologie et de Biologie Structurale (IPBS), France*

#### *Reviewed by:*

*Anca Dorhoi, Friedrich Loeffler Institute Greifswald, Germany Roberta Olmo Pinheiro, Oswaldo Cruz Foundation, Brazil*

#### *\*Correspondence:*

*Ankur Gupta-Wright<EMAIL_ADDRESS>David G. Russell<EMAIL_ADDRESS>
*† These authors have contributed equally to this work.*

#### *Specialty section:*

*This article was submitted to Microbial Immunology, a section of the journal Frontiers in Immunology*

*Received: 11 August 2017 Accepted: 15 September 2017 Published: 28 September 2017*

#### *Citation:*

*Gupta-Wright A, Tembo D, Jambo KC, Chimbayo E, Mvaya L, Caldwell S, Russell DG and Mwandumba HC (2017) Functional Analysis of Phagocyte Activity in Whole Blood from HIV/Tuberculosis-Infected Individuals Using a Novel Flow Cytometry-Based Assay. Front. Immunol. 8:1222. doi: 10.3389/fimmu.2017.01222*

The accurate assessment of immune competence through *ex vivo* analysis is paramount to our understanding of those immune mechanisms that lead to protection or susceptibility against a broad range of human pathogens. We have developed a flow cytometrybased, whole blood phagocyte functional assay that utilizes the inflammatory inducer zymosan, coupled to OxyBURST-SE, a fluorescent reporter of phagosomal oxidase activity. The assay measures both phagocytic uptake and the superoxide burst in the phagocyte populations in whole blood. We utilized this assay to demonstrate impaired superoxide burst activity in the phagocytes of hospitalized HIV-positive patients with laboratory-confirmed tuberculosis. These data validate the use of the assay to assess the immune competence of patients in a clinical setting. The method is highly reproducible with minimal intraindividual variation and opens opportunities for the rapid assessment of cellular immune competence in peripheral blood in a disease setting.

Keywords: phagocytosis, zymosan, inflammation, monocytes, neutrophils, HIV, tuberculosis, whole blood assay

#### INTRODUCTION

Bacterial killing assays in whole blood are well established and allow *ex vivo* assessment of immune function in patients, particularly in the context of assessing response to vaccines or evaluating new bactericidal therapies (1–4). The main read out of these assays is microbial killing measured *via* culture and colony counting, or fluorescence if reporter strain organisms are used.

Potential problems of these microbiological killing assays include difficulties in standardizing the number of microbes and their multiplication rate. The tendency of the microbes to aggregate inconsistently during assays may also result in misrepresentation of the actual numbers of microbes measured at the end of the assay. In addition, there are other factors that can result in microbial loss that are not dependent on the host immune response or antimicrobial therapy (5). Finally, because the read out is simply bacterial survival, these assays lack the ability to differentiate mechanisms of killing and the relative contributions of the different phagocyte lineages present in the blood.

Phagocytosis is an important mechanism in the microbial killing pathway of phagocytes. Deficiencies in phagocyte function likely predispose individuals to acquire or succumb to infectious diseases. An extensive range of dynamic assays of phagosome function have been developed that are capable of providing a broad range of physiological readouts from the phagosome (6, 7). These assays have mostly utilized inert beads derivatized with different fluorescent reporters and focused on human alveolar macrophages or murine bone marrow-derived macrophages in culture (8–10). By removing cells from whole blood or their usual tissue fluid, we are unable to assess the potentially important influence of soluble proteins such as cytokines, chemokines, or antibodies on phagocytosis and phagosomal behavior. We therefore sought to develop an assay using a reporter particle more suitable for probing phagocyte biology in whole blood. The assay is designed to provide reproducible, unbiased, realtime analysis of phagosomal function of immune cells and potentially identify patients with impaired immune responses.

We utilized zymosan derivatized with the oxidation-sensitive fluorescent reporter, OxyBURST-SE, to quantify phagosomal oxidase activity in peripheral blood phagocytes *in situ*. Zymosan is a preparation of a cell wall glucan from *Saccharomyces cerevisiae* that has been used as a model microbial particle in immune assays for over half a century (11). Zymosan is highly mannosylated and linked to β-glucan, making it susceptible to phagocytosis by monocytes, polymorphonuclear leukocytes, and macrophages through various receptors, including C-type lectin receptors such as dectin-1 and mannose receptors (12, 13). Phagocytosis of zymosan can occur independent of opsonization, of which complement factor 3 (C3) predominates with immunoglobulin G (IgG) being of minor importance (14). Zymosan also stimulates an inflammatory cytokine response *via* toll-like receptors (TLR) 2 and 6, although activation of these receptors is not required for internalization by phagocytes (12). We had demonstrated previously how inert particles coupled to OxyBURST-SE can be used to quantify the superoxide bust of murine macrophages *in vitro* (15).

Superoxide burst is one of the key enzymatic activities involved in killing microbes during the process of phagocytosis. The generation of oxygen radicals *via* nicotinamide adenine dinucleotide phosphate (NADPH) oxidase leads to the production of noxious compounds such as hydrogen peroxide with potent antimicrobial activity (16, 17). Superoxide burst's importance is clearly demonstrated by the greatly increased risk of bacterial, fungal, and mycobacterial infection in patient with chronic granulomatous disease due to mutations in NADPH oxidase (18). It has also been shown to be suppressed in individuals with HIV infection (19) and by *Mycobacterium tuberculosis* (TB) infection *in vitro* (20).

In this study, we report the application of this novel reporter platform to quantify the phagocytic and superoxide burst functions of phagocytes in whole blood obtained from individuals in a clinical setting. First, we detail the information generated by application of the assay in whole blood from healthy controls. We then present data showing the utility of this assay in demonstrating the perturbation of phagocyte function in the blood from HIV- and TB-coinfected patients in Malawi.

#### MATERIALS AND METHODS

#### Study Population

Adult patients with HIV and tuberculosis coinfection (HIV-TB) were recruited as part of a sub-study examining immune responses in the Malawi arm of the rapid urine-based screening for TB to reduce AIDS-related mortality in hospitalized patients in Africa (STAMP) (21). Healthy HIV-negative adults with no evidence of active TB were also recruited as controls. 5 ml of blood was collected from both patients and controls in sodium heparin tubes. All samples were processed and analyzed by flow cytometry at the Malawi-Liverpool-Wellcome Trust Clinical Research Programme in Blantyre, Malawi within 2 h of blood draw. The study has been approved by the London School of Hygiene & Tropical Medicine Research Ethics Committee and the College of Medicine Research Ethics Committee, Malawi.

#### Zymosan Reporter Particles

To quantify both phagocytic activity and the magnitude of the superoxide burst we utilized zymosan particles coupled to both a calibration fluorochrome (Alexa Fluor 405-SE, Invitrogen) and an oxidation-sensitive fluorescent reporter (OxyBURST® Green H2DCFDA-SE, Invitrogen). Zymosan reporter particles were prepared by washing 6 mg of zymosan (Sigma-Aldrich) three times in 1× phosphate-buffered saline (PBS) by centrifugation at 10,000 rpm for 1 min. Particles were resuspended in 950 µl coupling buffer (0.1 M boric acid to pH 8.0 with NaOH) containing 10 µl of 25 mg/ml OxyBURST-SE/DMSO stock solution and 5 µl of 5 mg/ml Alexa Fluor 405-SE/DMSO solution. The particles were mixed well and incubated on a tube rocker in the dark for 1 h at room temperature and washed with 1 ml of coupling buffer. The 1 h coupling with OxyBURST-SE and calibration fluorochrome was repeated twice. Finally, particles were washed three times with PBS and stored in 1 ml of PBS containing 0.01% sodium azide in the dark at 4°C generating a final stock concentration of approximately 5 × 106 particles/ml.

#### Whole Blood Assay

Zymosan reporter particles were prepared for the whole blood assay by washing 50 µl of stock Zymosan particle suspensions three times with 1 ml of RPMI-1640 to remove sodium azide and resuspended in 250 µl RPMI-1640 to give a 1:6 dilution and a final concentration of approximately 8 × 105 particles/ml.

Whole blood was diluted 1:1 with warm RPMI-1640. 20 µl of washed and diluted reporter particles (containing approximately 2 × 104 particles) were added to 1 ml of diluted blood and incubated at 37°C with rocking to ensure particles and cells remain in suspension. Diluted blood without zymosan reporter particles was also processed in parallel as control. Phagocytosis of zymosan reporter particles and superoxide burst was assessed at 10, 30, 60, 90, and 180 min after the addition of reporter particles.

100 µl of diluted blood was harvested from the zymosan reporter and biological control tubes 10 min before each time point for cell surface staining (as phagocytosis continues during cell surface staining of live cells). Once harvested, the diluted blood was stained with appropriately titrated concentrations of antibodies (anti-CD45 PerCP 1:33, anti-CD66b APC 1:50, and anti-CD14 PE-Cy7 1:100; all from BioLegend) for 10 min. Biological activity was arrested, red blood cells were lysed, and leukocytes fixed by adding 3 ml of BD FACS lysing solution (BD Biosciences), containing formaldehyde and diethylene glycol, to each tube and incubating at room temperature for 10 min. The cells were washed once with 1× PBS by centrifugation at 500 *g* for 10 min then resuspended in 500 µl 1× PBS. Counting beads (Countbright, Life Technologies) were added per the manufacturer's instruction before acquisition on a CyAn ADP flow cytometer (Beckman Coulter, USA). The phagocytosis assay was performed in triplicate on the whole blood samples from healthy, HIV-negative adults. Data were analyzed using FlowJo version 10 (Treestar, USA).

In addition to the zymosan reporter assay, for HIV/TBcoinfected patients, immunophenotyping of monocytes in fresh whole blood was undertaken to investigate the association between monocyte phenotype and phagocytosis. In brief, 100 µl of fresh whole blood was stained with anti-CD45 Pacific Orange (Invitrogen), anti-HLA-DR PE-Cy7, anti-CD14 PE, and anti-CD16 FITC (all from BioLegend) for 10 min. Red blood cells were lysed, and leukocytes fixed with BD FACS lysing solution, washed once with 1× PBS by centrifugation at 500 *g* for 10 min then resuspended in 300 µl PBS for flow cytometry acquisition.

#### Electron Microscopy (EM)

In parallel, 2 ml of whole blood from a healthy HIV-negative control was incubated with approximately 8 × 104 zymosan reporter particles to confirm the zymosan particles were internalized by whole blood phagocytes. White blood cells were harvested after 10, 60, and 180 min by centrifugation at 500 *g* for 10 min and carefully pipetting out the buffy coat layer in buffered glutaraldehyde fixative solution (2.5% glutaraldehyde in 0.1 M sodium cacodylate, 5 mM CaCl2, 5 mM MgCl2, 0.1 M sucrose, pH 7.2). The samples were processed and stained for EM as described previously (22).

#### Calculations and Statistical Analysis

The proportion of cells that had phagocytosed reporter particles was calculated based on expression of calibration fluorochrome, and absolute cell numbers calculated using counting beads. An "activity index" of phagocytosis and superoxide burst was calculated by subtracting the median fluorescence intensity of the negative cells from the positive cells, and dividing this by two times the robust SD of the negative cells (23). This method accounted for variations in auto fluorescence between cells from different individuals.

Statistical analysis was performed using GraphPad Prism 7 (GraphPad Software, USA) and Stata 11 (StataCorp, USA). Peak activity index (AI) was calculated and mean AI was compared between groups. The AI at each time point was also used to calculate the area under the curve. Means were compared using paired *t*-tests and median using Wilcoxon rank-sum.

#### RESULTS/DISCUSSION

#### Zymosan Uptake by Whole Blood Phagocytes

We used whole blood from four healthy HIV-negative controls to measure phagocytosis and superoxide burst of phagocytes *ex vivo* using zymosan-reporter particles. We first sought to determine the kinetics of zymosan uptake by whole blood phagocytes. The flow cytometry gating strategy to identify neutrophils and monocytes is outlined in **Figure 1**. Cells that had phagocytosed zymosan-reporter particles were identified and quantified through measurement of the calibration fluor, Alexa Fluor 405.

Zymosan particles were avidly internalized by both neutrophils and monocytes in blood from healthy controls. Uptake was rapid, with a mean of 26% of neutrophils phagocytosing the particles compared with 12% of monocytes by 30 min (**Figure 2A**). The proportion of neutrophils phagocytosing zymosan did not increase substantially between 30 and 180 min, whereas the percentage of monocytes associated with zymosan-reporter particles increased gradually during the assay. This pattern of uptake was consistent across all healthy controls.

The uptake of zymosan reporter particles by both monocytes and neutrophils is dose dependent as shown in the dose–response curve generated for 0.5 × 104 –8 × 104 zymosan particles/ml (**Figure 2B**). The abundance of the phagocytic cells in whole blood also influences the overall proportion of cells phagocytosing zymosan particles (**Figure 2C**). The higher the concentration of cells, the lower the proportion of cells carrying the zymosanreporter signal, shown for both neutrophils and monocytes (**Figures 2D,E**). This relationship persists throughout the assay and demonstrates the importance of the phagocyte to particle ratio in the kinetics of phagocytosis. Relying solely on internalization of particles to assess phagocytic function is a potential limitation of the assay, as the magnitude of phagocytosis may be influenced by a function of cell concentration and/or cell to particle ratio, rather than cellular deficiencies in phagocytic capacity.

Electron microscopy of white blood cells from a healthy control whole blood incubated with zymosan particles demonstrates phagocytosis of zymosan particles by peripheral blood phagocytes (**Figure 3**). The EM images support the assumption that the zymosan reporter signal detected by flow cytometry originates from phagocytosis rather than the association of zymosan particles with the phagocyte surface. Almost without exception, the zymosan particles were observed inside the phagocyte.

#### Cell Loss Associated With Zymosan

To examine the effect of the zymosan particles on cell loss, we compared the samples containing zymosan reporter particles and control samples from the same healthy individuals. The mean concentration of neutrophils and monocytes declined during the assay more rapidly in the presence of zymosan than in control samples, with the largest decline occurring between 90 and 180 min (**Figures 4A,B**).

Furthermore, the concentration of neutrophils associated with zymosan-reporter signal peaked at 60 min, followed by a decline (**Figure 4A**). By contrast, the concentration of zymosanassociated monocytes plateaus at 30 min (**Figure 4B**). However, in both cell types the peak in zymosan uptake coincided with cell loss, suggesting that zymosan plays a role in inducing cell death. This is also supported by increased cell loss at higher concentrations of zymosan in the assay (**Figure 2B**).

These observations are consistent with neutrophil and monocyte biology. Neutrophils are known to have a short halflife *in vitro*, estimated to be 6–12 h, and do not proliferate (24). Programmed cell death of neutrophils occurs rapidly following

illustrated is from one representative healthy volunteer. (B) Zymosan-induced superoxide burst activity in neutrophils and monocytes in a healthy control. The OxyBURST fluorescence increases after intraphagosomal oxidation of the zymosan-reporter particles (Alexa Fluor 405-labeled) after 10 and 60 min compared with control sample with no zymosan-reporter particles. (C) Overlay histogram demonstrating the shift in fluorescence of cells with zymosan reporter particles due to oxidation after 10 min (red), 30 min (orange), and 60 min (blue) compared with cells without zymosan-reporter particles (green) for both neutrophils and monocytes.

phagocytosis of inflammatory particles, and reactive oxygen species may be important triggers for induction of apoptosis (20). In neutrophils that have not phagocytosed zymosan particles, activation *via* direct binding of zymosan to TLR2 and TLR6 or in response to inflammatory cytokine and chemokine production may also contribute to cell death (12). By contrast, monocytes have an estimated half-life of <20 h *in vivo*, although this may be shorter *ex vivo* (25). Monocytes also undergo programmed cell death, unless they migrate to tissues and undergo differentiation into tissue macrophages (26). However, in contrast to neutrophils, inflammatory cytokine production and stimulation *via* TLR2 can promote survival by blocking programmed cell death (27). This may explain why monocytes that had phagocytosed zymosan reporter particles did not substantially decrease in number during the assay.

#### Phagocytosis and Superoxide Burst

Superoxide burst activity was measured at 10, 30, 60, 90, and 180 min by comparing fluorescence of cells that had internalized zymosan reporter particles (calibration fluor-positive cells) with the cell population without zymosan (calibration fluor-negative cells) through measurement of the OxyBURST, superoxide sensor signal. The proportion of cells and intensity of superoxide reporter fluorescence increased over the time course of the assay in both

FIGURE 2 | Phagocytosis of zymosan reporter particles by monocytes and neutrophils. (A) Proportion of cells that have phagocytosed zymosan over time using blood from healthy individuals incubated with approximately 2 × 104 zymosan particles/ml. Data represent mean values, and the error bars indicate SEM. Concentration of neutrophils (B) and monocytes (C) that have internalized zymosan reporter particles over time, with varying concentrations of particles. Relationship between the proportion of cells internalizing zymosan reporter particles (at an approximate concentration of 2 × 104 zymosan particles/ml) and absolute cell concentration for neutrophils (D) and monocytes (E) after 60 min. Each data point represents one sample from each individual done in triplicate. Four healthy volunteers in triplicate are shown in panels (A,D,E), and one health volunteer is shown in panels (B,C).

monocytes and neutrophils (**Figures 1B,C**). The intensity of the calibration fluorochrome signal did not increase over time, suggesting the increase in superoxide reporter signal was not due to cells internalizing greater numbers of zymosan-reporter particles but was specific to the oxidase activity (**Figure 1B**).

Both peripheral blood monocytes and neutrophils showed rapid oxidation within 30–60 min (**Figures 4C,D**). The kinetics of oxidation in neutrophils and monocytes were similar to macrophages in other studies, with rapid oxidation before an equilibrium being reached, which likely represents cessation of NADPH oxidase activity (6, 15).

When the concentration of zymosan reporter particles was varied, the AI remained constant despite the concentration and proportion of cells taking up zymosan changing. This indicates the assay is able to measure physiological changes in the intensity and duration of phagocytosis and superoxide burst within the phagosome at an individual cell level. This is a significant advance over existing assays, which measure the extracellular accumulation of products of oxidation that is dependent on the summation of phagocytosis and superoxide burst (28). Moreover, because this assay has cellular resolution, the relative contribution of the different phagocyte subsets can be accurately measured. We have also demonstrated that the assay is reproducible with minimal intraindividual variation.

Assays using OxyBURST coupled to IgG coated beads have previously been used to investigate oxidation within macrophage phagosomes (6, 15), and more recently in whole blood (29). The current assay exploiting zymosan as a reporter particle is an

FIGURE 3 | Assessment of phagocytosis of zymosan reporter particles by electron microscopy. An electron micrograph illustrating zymosan particles (Z) inside a neutrophil 60 min post incubation of the reporter particles with whole blood. A red blood cell (R) can be seen to the right of the neutrophil. This image is representative and indicates that the zymosan particles are effectively internalized by cells in suspension. The scale bar = 1 µm.

important addition to the range of phagocyte functional assays and offers considerable practical and technical advantages in the functional interrogation of whole blood direct from human subjects of interest.

#### Assessment of Whole Blood Phagocyte Function in Patients with HIV/TB Coinfection

The zymosan reporter assay was performed on blood samples obtained from 18 hospitalized HIV-positive patients with laboratory confirmed TB disease to compare phagocytic and superoxide respiratory burst activity in the phagosome between patients and healthy, HIV-negative, controls. The HIV/TB patients had a mean age of 41.4 years, a median CD4 cell count of 108.5 cells/mm3 and 13/18 were taking antiretroviral therapy at the point of hospital admission. The HIV/TB-coinfected patients demonstrated marked variation in phagosomal oxidation activity compared with healthy controls. The kinetics were similar to healthy controls with peak activity occurring at 30 min, although overall mean intensity of superoxide burst was significantly reduced throughout the assay (paired *t*-test, all *p* < 0.0001) (**Figure 5A**).

There was also a strong association between increased monocyte superoxide burst activity and the presence of a higher proportion of "classical" CD14++CD16<sup>−</sup> monocytes (**Figure 5B**, linear regression coefficient 0.0014, 95% CI 0.0005–0.0024, *p* = 0.006). This association is consistent with the suggestion that classical monocytes are thought to specialize in phagocytosis compared with other monocyte subsets (30). However, the superoxide activity in monocytes was not related to the overall concentration of monocytes in patient's blood (linear regression slope 0.0002, 95% CI −0.0002 to 0.0005, *p* = 0.19), supporting the contention that the superoxide AI was not simply a function of phagocyte abundance.

These data demonstrate that the whole blood assay with zymosan reporter particles is a robust tool for assessing phagocyte function in a clinical setting. The time required to run the assay once the reporter particles have been made is minimal, with the processing of the sample through to acquisition by flow cytometry taking less than 4 h. We also demonstrated this assay can show marked differences between individuals and groups of patients based on clinical phenotype. It is interesting to note that the reduced superoxide burst in the phagocytes from HIV/ TB-coinfected individuals observed in this study is consistent with a recent report of impaired innate immune function of monocytes from HIV/TB-coinfected patient cohort in South Africa (31).

#### CONCLUDING REMARKS

We present a new method for studying whole blood phagocyte functional capacity *ex vivo*. This technique uses fluorescenttagged zymosan-reporter particles and whole blood, preserving, at least in part, the physiological *in vivo* conditions. It offers several advantages over standard microbiological killing assays because of its speed and simplicity, and its increased resolution whereby cellular responses such as phagocytic capacity and superoxide burst, can be quantified at the level of the individual cell.

We have demonstrated that the assay can be used to characterize immune function and to detect perturbation of cellular function in patients with severe immunological impairment (in HIV/TB-coinfected individuals). This assay is easily adaptable to standard immunological assays based on cell surface marker expression measured by flow cytometry and has the capacity to provide direct functional readouts of immune cell activities. Previously, we have used inert reporter particles to measure rates of phagosomal acidification, intraphagosomal proteolytic and lipolytic activities, as well as superoxide burst in tissue macrophages in culture. These activities are differentially modulated by immune status and infection (7–9). The use of zymosan as an alternative, biologically active carrier particle for whole bloodbased assays brings these complex biological readouts into a clinical setting for functional interrogation of patient-derived samples linked to disease status.

#### ETHICS STATEMENT

The study was carried out in accordance with the recommendations from the London School of Hygiene & Tropical Medicine

#### REFERENCES

Research Ethics Committee and the College of Medicine Research Ethics Committee, Malawi, with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki.

### AUTHOR CONTRIBUTIONS

All the authors contributed to the analysis and interpretation of data and preparation of the manuscript. All the authors have approved the final article.

### FUNDING

This work was supported by the Royal College of Physicians London JMGP fellowship and the Joint Global Health Trials Scheme (grant number MR/M007375/1) to AG-W, the Wellcome Trust (088696/Z/09/Z) to HCM and (105831/Z/14/Z) to KCJ, the Bill and Melinda Gates Foundation awards (OPP1125279) to HCM and (OPP1156451) to DGR, and the US National Institutes of Health awards AI118582, AI089683, and AI134183 to DGR. A Strategic award from the Wellcome Trust supports the MLW Programme.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Gupta-Wright, Tembo, Jambo, Chimbayo, Mvaya, Caldwell, Russell and Mwandumba. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Defective Phagocytic Properties of Hiv-infected Macrophages: How Might They Be implicated in the Development of invasive *Salmonella* Typhimurium?

#### *Gabrielle Lê-Bury1,2,3 and Florence Niedergang1,2,3\**

*<sup>1</sup> INSERM, U1016, Institut Cochin, Paris, France, 2CNRS, UMR 8104, Paris, France, 3Université Paris Descartes, Sorbonne Paris Cité, Paris, France*

Human immunodeficiency virus type 1 (HIV-1) infects and kills T cells, profoundly damaging the host-specific immune response. The virus also integrates into memory T cells and long-lived macrophages, establishing chronic infections. HIV-1 infection impairs the functions of macrophages both *in vivo* and *in vitro*, which contributes to the development of opportunistic diseases. Non-typhoidal *Salmonella enterica* serovar Typhimurium has been identified as the most common cause of bacterial bloodstream infections in HIVinfected adults. In this review, we report how the functions of macrophages are impaired post HIV infection; introduce what makes invasive *Salmonella* Typhimurium specific for its pathogenesis; and finally, we discuss why these bacteria may be particularly adapted to the HIV-infected host.

### *Edited by:*

*Christel Vérollet, UMR5089 Institut de Pharmacologie et de Biologie Structurale (IPBS), France*

#### *Reviewed by:*

*Aldo Tagliabue, Istituto di Ricerca Genetica e Biomedica (CNR), Italy Elsa Anes, Universidade de Lisboa, Portugal*

*\*Correspondence: Florence Niedergang<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Microbial Immunology, a section of the journal Frontiers in Immunology*

*Received: 03 November 2017 Accepted: 28 February 2018 Published: 23 March 2018*

#### *Citation:*

*Lê-Bury G and Niedergang F (2018) Defective Phagocytic Properties of HIV-Infected Macrophages: How Might They Be Implicated in the Development of Invasive Salmonella Typhimurium? Front. Immunol. 9:531. doi: 10.3389/fimmu.2018.00531*

Keywords: macrophages, HIV, *Salmonella*, phagocytosis, opportunistic diseases

# CLEARANCE AND ACTIVATION CAPACITY OF MACROPHAGES

Although macrophages are different in the different tissues and organs in which they reside, they can all be characterized by their strong capacity to internalize and degrade particles in phagolysosomes. Transcriptomic analysis has pointed to unique gene expression profiles in response to pathogens components (1). One obvious functional set of genes shared by all phagocytes encodes for the components of lysosomes, such as the vacuolar ATPase H<sup>+</sup> pump and lysosomal hydrolases. Subtle differences can be revealed when comparing the clearance capacities of polymorphonuclear neutrophils, macrophages or dendritic cells. Clearance is the hallmark of neutrophils and macrophages, while dendritic cells have been reported to be milder with internalized material, with some material remaining undegraded (2–5). In this review, we will focus on macrophages, as they are target cells for both the human immunodeficiency virus type 1 (HIV-1) and *Salmonella* pathogens.

Phagocytosis begins with the clustering of receptors that are engaged by ligands present on the surface of the target particle. Many types of receptors can be implicated in the recognition step, regulating the fate of the internalized material. Phagocytic receptors can be subdivided into receptors that bind to opsonins, like the immunoglobulins and complement, and receptors that bind to non-opsonins. The latter interact with molecular groups on the surface of the target particle or pathogen, including sugars, lipids, and polypeptides that are referred to as pathogen-associated molecular patterns (6, 7). Early signaling from surface receptors leads to the polymerization of actin that drives plasma membrane deformation and the formation of a closed phagosome (8, 9). For large targets to be efficiently internalized, membrane remodeling is essential, relying on the focal delivery of intracellular compartments including recycling endosomes (10–12). The large GTPase dynamin2 is crucial for phagosome sealing (13).

Once closed, the phagosome evolves very much like smaller endocytic compartments, undergoing a series of fusion and fission events with the compartments of the endocytic pathway (14, 15). This process, called phagosome maturation, is accompanied by the dynein-mediated movement of the phagosome along microtubules (16, 17). The microtubule network is crucial for the phagosome to reach the lysosomes positioned at the center of the cell and to efficiently mature into a phagolysosome. Indeed, it has been demonstrated that the loading of dyneins at the plus ends of microtubules by the plus-end binding protein, EB1, is critical for phagosome maturation (18).

Early phagosomes harbor markers of the early endosomes such as Rab5 and its effector, the early endosome antigen 1 (EEA1) (19). Other effectors of Rab5 are the class III phosphatidylinositol 3 kinase human vacuolar protein-sorting 34 that generates phosphatidylinositol 3-phosphate [PI(3)P] (20). It has been demonstrated that PI(3)P is important after phagosome completion, for its maturation (21). EEA1 carries a FYVE domain that binds to PI(3)P, a zinc finger that binds to Rab5 and regions responsible for multimerization. EEA1 also binds to the t-SNARE Syntaxin-13, which is important for membrane fusion (22). Rab5 exchange factors, including Rabex-5, Rin1, and Gapex-5, coordinate Rab5 activation and microtubule dynamics (23).

Acquisition of Rab7 is still considered to be a hallmark of late phagosomes, although a choreography of Rab proteins has been shown to be recruited during phagocytosis (24). The products of PI3K are involved in the dissociation of Rab5, but are not essential for the recruitment of Rab7 on the phagosome (25). Data in yeast have shown that the proteins Mon1 and Ccz1 serve as a Rab7 exchange factor (26). Phagosomes undergo fusion with late endosomes and lysosomes *via* a Soluble NSF attachment protein receptor (SNARE) mediated process. It has been demonstrated that Syntaxin 7 and Syntaxin 8, with VAMP7 and VAMP8, are involved in phagosome-lysosome fusion (27). The vpsC–homotypic protein sorting (HOPS) complex that mediates the transition from Rab5 to Rab7 endosomes could play a similar function in phagosome maturation. The complex is composed of Vps11, Vps16, Vps18, Vps33, Vps39, and Vps41. In yeast, Rab7 is activated by Vps39. The Vps41 protein is a key component of the HOPS complex as it is required for the stabilization of the HOPS complex on the endosomal membrane before fusion with the vacuole. Regulation by the p38 MAP kinases of Vps41 has recently been highlighted as an explanation for the differential trafficking of virulent LPS of *Coxiella burnetii* (28). This type of regulation might also be implicated in phagosome maturation in macrophages. Rab7 and Arl8 orchestrate both microtubuledependent transport of late endosomes/lysosomes and their fusion with endosomes, autophagosomes, and phagosomes. Both proteins are important for lysosome tubulation in macrophages (29). Rab7-interacting lysosomal protein and the long splicevariant of oxysterol-binding protein related-protein 1 (ORP1L) function together to link phagosomes to the microtubule motor dynein/dynactin (30, 31). Arl8 has been shown to control phagosome maturation and bacterial killing (32). It has also been demonstrated that Arl8 plays an important role in phagosome maturation in *Caenorhabditis elegans* (33). Arl8 connects lysosomes to kinesins through SifA and kinesin interacting protein (34), in particular, to kinesins 1 and 3, controlling lysosomal positioning (35). Some effectors, like PLEKHM1, act as dual effectors for Rab7 and Arl8 to promote cargo delivery to lysosomes (36) and may also play a role during phagosome maturation.

Acidification occurs gradually while the compartment matures into a phagolysosome, which is important for the optimal activity of hydrolytic enzymes delivered by late endosomes and lysosomes. Acidification reaches a pH of 5.5 in the late phagosome, due to the acquisition of proton pumping vATPases. Negatively charged chloride ions may enter the compartments to compensate for the proton influx, although this role has not been established experimentally. However, the depletion of luminal cations, Na<sup>+</sup> and K<sup>+</sup>, during maturation has been established (37). Anion and luminal cation exchange may serve to maintain lysosome osmolarity and volume during the acidification steps.

Microbicidal activity in the phagolysosome depends both on hydrolases and on the generation of reactive oxygen and nitrogen species (ROS and RNS, respectively) (38). The NADPH oxidase is acquired at the early stages of phagosome maturation. Its activity is complemented by the inducible NO synthase, iron scavengers, and transporters, as well as lysozymes, lipases and proteases, such as the cathepsins. These species are delivered to phagosomes and directly contribute to killing. Their activities can be modulated by cell activation and the concomitant signaling pathways that are initiated downstream of surface receptors in complex regulatory loops. For instance, the interaction between the RUN domain Beclin-1 interacting cysteine-rich-containing (RUBICON) protein and the NADPH oxidase upon TLR stimulation forms a feedback loop with the cascade signaling to cytokine production (39).

Finally, when degradation is incomplete, antigens derived from the internalized material are presented on the major histocompatibility complex molecules (Class I or II), especially in dendritic cells. Signaling pathways are activated and the phagosome itself can be considered a signaling platform. Together, these events lead to the production of cytokines and inflammatory mediators, which can be modulated depending on the surface receptors engaged by the cargo (40, 41).

#### DYSREGULATION OF MACROPHAGE PHAGOCYTOSIS BY HIV INFECTION

The initial detection of the acquired immunodeficiency syndrome (AIDS) epidemic in 1981 began with reports of an unusual syndrome in which previously healthy young males were presenting with diseases such as Kaposi's sarcoma, cytomegalovirus pneumonia, and *Pneumocystis carinii* pneumonia, previously described in immunocompromised patients (42). The syndrome was then named AIDS. Indeed, the principal effect of the virus is to decrease the immune defenses of infected individuals, leading to the appearance of opportunistic diseases and tumors. Invasive pneumococcal and oral candidosis presented early in HIV-infected patients, especially in Africa, together with nontyphoidal *Salmonella* infections, which will be the focus of the following section in this review.

The introduction of highly active antiretroviral therapy (HAART) has radically altered the incidence of AIDS, initially in developed countries, and later on, worldwide. However, even with HAART, which results in undetectable plasma HIV loads, HIV transcription persists in reservoirs, such as long-lived memory T cells, phagocytic cells, as well as other cell types in various tissues (43).

Already in the 1980s, Crowe et al. reported that macrophages are targets for HIV-1 and may act as major reservoirs of virus (44, 45). It has since been established that there is some specificity in the infection cycle of HIV-1 in macrophages (46–49) and see Ref. (50) for a recent review in the same topic.

The group of Crowe has performed pioneering work in analyzing the phagocytic capacity of blood monocytes from a small sample of the Sydney Blood Bank Cohort or *in vitro* differentiated and infected macrophages. They studied phagocytosis of apoptotic neutrophils, a *Mycobacterium avium* complex, *Candida albicans*, *Toxoplasma gondii*, and IgG- or complement-opsonized targets (51–55). The data suggested that phagocytosis by monocytes from WT HIV-1-infected individuals was impaired, whereas the phagocytic capacity of the phagocytes from the Δ*nef* HIV-1-infected subjects was not. Interestingly, no correlation was found between the level of inhibition of phagocytosis and the viral load or the CD4 counts (52). The authors reported an increased level of basal F-actin in HIV-infected cells, which could account for a defective capacity of the actin cytoskeleton to efficiently remodel during phagosome formation (**Figure 1A**).

Figure 1 | Modified phagocytosis in HIV-infected macrophages. Left panel—Once HIV infection of a macrophage is established, intracellular trafficking is rerouted to the virus-containing compartment (VCC). In non-infected macrophages, phagocytosis is initiated by the binding of phagocyte surface receptors to ligands present on the microorganism or to the opsonizing molecules that coat the target particle. In HIV-infected cells, surface receptors (e.g., FcR) can be downregulated (A). After binding, a cascade of signaling events leads to actin polymerization and engulfment of the particulate material in a closed compartment termed, the phagosome. The inhibition of phagocytosis in HIV-infected macrophages was related to perturbation of F-actin and cAMP production. The viral factor Nef further reduces the efficiency of phagosome formation *via* its interaction with the AP1 adaptor protein, reducing the focal delivery of intracellular compartments (A). In non-infected macrophages, the phagosome matures into a degradative compartment called phagolysosome. This occurs after fusion and fission with various endocytic compartments, and the phagolysosome migrates along microtubules. However, in HIV-infected macrophages, the viral factor Vpr inhibits phagosome maturation and centripetal movement of the phagolysosome toward the nucleus. Part of the intracellular trafficking, such as the EHD3 recycling machinery, is rerouted to the VCC (B). In addition, viral infection inhibits macrophage late events and responses such as cytokine production (C). Right panel—Primary human macrophages were infected with HIV-1ADA for 8 days before incubation with IgG-opsonized sheep red blood cells (SRBCs) for 60 min at 37°C. They were fixed, permeabilized, and labeled with anti-p24 followed by Alexa488-anti-goat IgG (upper line), AMCA-anti-rabbit IgG to detect the total SRBCs (second line), anti-LAMP1 followed by Cy3-anti-mouse IgG (third line), and anti-tubulin followed by Cy5-anti-human IgG (not shown). Merged images (lower line) show p24 in green, SRBCs in blue, LAMP1 in white, and microtubules in red. Z stacks of wide field fluorescent images were acquired, deconvoluted, and treated with ImageJ. Bar, 10 µm.

Some HIV-1 induced receptor downregulation was observed in primary human macrophages (56), as in alveolar macrophages for mannose receptors (57). The group of Crowe also pointed to a crucial role of the Nef viral protein in impairing phagocytosis in infected blood monocytes *in vivo*, a phenotype that they did not observe in monocyte-derived macrophages-infected *in vitro*. Nef was reported to downregulate many surface receptors and markers from the surface of treated model cells (58, 59), as well as CD36 on macrophages, which may be related to defective phagocytosis of inert particles and killed bacteria in Nef-treated cells (60). Other reports showed that HIV-1 infection did not induce any decrease in the surface expression of phagocytic receptors (52, 61). The impact of *in vitro* infection of macrophages with HIV-1 on various types of phagocytosis was analyzed. The focal delivery of endosomal compartments at the site of phagocytosis was impaired in a Nef-dependent manner, probably due to Nef interaction with the adaptor complex, AP1, on endosomes (61) (**Figure 1A**). This endosomal delivery was shown to be required for efficient phagocytosis of large particulate material (11, 62), and interestingly, to be controlled by the NF-κB signaling protein Bcl10 (63). Other molecular defects reported in HIV-infected macrophages include elevated intracellular cAMP levels (64) and decreased expression of the common gamma chain (65) (**Figure 1**). Of particular significance, defective phagocytosis was also reported in the population of small alveolar macrophages in the lung of HIV-infected patients (66).

The later steps of phagosome maturation were also reported to be impaired in HIV-infected macrophages, with early reports showing that the intracellular replication of live *T. gondii* was enhanced in HIV-infected macrophages. Interestingly, treatment of macrophages with interferon gamma, a known activator of these cells, decreased parasite replication, but did not control parasite levels (53). An assay developed in the laboratory of Russell to monitor the superoxide burst in phagocytes by flow cytometry was used to analyze whole blood samples of HIV/ tuberculosis-infected individuals (67). The authors were able to demonstrate an impaired superoxide burst activity in the phagocytes of coinfected patients. Using similar tools together with other assays, it was determined that Nef was not crucial to the *in vitro* mediation of the phagosome maturation defect in HIVinfected macrophages, while, unexpectedly, the viral protein Vpr, was (**Figure 1B**) (18). Vpr perturbs the microtubule dynamics, the localization of the plus-end microtubule binding protein EB1 and therefore, the positioning of the dynein motors necessary for driving phagosomes to the cell center. In addition, the viral infection of macrophages relies on the budding of newly enveloped viral particles in virus-containing compartments (VCCs), which presumably requires the recruitment of large amounts of membrane. Part of the intracellular endocytic machineries are de-routed toward the VCCs in HIV-1-infected macrophages, as is the case for the EHD3 sorting protein (18). It is therefore probable that many of the intracellular trafficking pathways are altered in virus-infected cells, as a side effect of viral particle production. This may benefit many opportunistic pathogens in a non-specific manner.

Similarly, signaling to initiate cell activation and cytokine production is altered in HIV-infected cells (**Figure 1C**). Placental blood mononuclear cells purified from HIV-infected mothers constitutively secrete more IL-1β and IL-6 and have more IL6, IL1β, and TNF-α mRNA; however, the high basal rates of secretion were associated with a lower response to stimulation with LPS (68). Reduced cytokine production was also observed in HIV-infected macrophages that were triggered for receptormediated phagocytosis or infected with bacteria (18). Many proinflammatory cytokine signaling pathways rely on the activation of the NF-κB pathway. The latter is transiently activated during the activation of transcription *via* the HIV promoter or the long terminal repeats. This may result in inadequate subsequent activation when the cells are subjected to a secondary trigger. Reduced intracellular protein levels of FcRγ, the signaling adaptor protein and chaperone required for FcγRI and III expression and function, were reported (69). Inhibition of subsequent downstream phosphorylation of Hck and Syk tyrosine kinases was observed in HIV-infected monocyte-derived macrophages undergoing Fcγ receptor-mediated phagocytosis.

### UNIQUE INTRINSIC PROPERTIES OF INVASIVE NON-TYPHOIDAL SALMONELLAE (iNTS)

In as early as 1990, non-typhoidal salmonellae (NTS) were confirmed as HIV-related pathogens in sub-Saharan African adults (70). Later, NTS bacteremia has become a common and recurrent illness among susceptible African children and HIV-infected adults (71). This bloodstream infection, in African, HIV-infected adults, was reported to have high mortality (47%) and recurrence (43%) rates (72), due to recrudescence and reinfection (73). The bacteremia may be due to both *Salmonella enterica* serovar Typhimurium and *Salmonella enterica* serovar Enteritidis (74).

*Salmonella enterica* is a Gram-negative bacterium that causes enteric diseases. The species, *S. enterica,* includes typhoidal and non-typhoidal *Salmonella* and comprises a large number of serovars. *S. enterica* serovars Typhi and Paratyphi cause typhoid and paratyphoid fevers, respectively. The pathogens penetrate through the intestinal mucosa, producing bacteremia and lodge in the macrophages of the reticuloendothelial system. The remaining serovars normally lead to a self-limiting diarrheal disease in healthy individuals, but some NTS, such as *Salmonella* Typhimurium, can cause bloodstream infection in immunocompromised adults (75). Thus, iNTS have emerged as a prominent cause of bacteremia in African individuals with an associated case fatality of 20–25% (76).

Multilocus sequence typing (MLST) analysis of numerous isolates of *Salmonella* Typhimurium from Malawi and Kenya revealed new sequence type variants of *S.* Typhimurium associated with iNTS in sub-Saharan Africa. This dominant regional genotype, MLST group ST313, presents several genetic differences compared with other strains of this serotype, such as NTS ST19 (77).

In addition, whole-genome sequence-based phylogenetic methods revealed that the majority of ST313 isolates fell within two closely related, clustered phylogenetic lineages: lineage I, with A130 as a hallmark strain; and lineage II, with D23580 as a hallmark strain (78). These lineages are distinct from other *S.* Typhimurium lineages due to their distinct metabolic profiles (79) and antibiotic resistance (77). Indeed, Okoro et al. observed that isolates from lineage II appeared after the use of chloramphenicol for the treatment of iNTS disease, suggesting a clonal replacement of isolates from lineage I, by those from lineage II influenced by antibiotic usage. In addition, the authors estimated that lineage I and lineage II appeared independently, ~52 and ~35 years ago, respectively, and then developed with the HIV pandemic. Thus, Okoro et al. propose that iNTS disease, in sub-Saharan Africa, is caused by highly related *Salmonella* Typhimurium lineages that may have developed in immunosuppressed populations and following antibiotic treatment (78).

Host-adapted *Salmonella* serovars that cause invasive disease such as *S*. *enterica* Typhi and *S. enterica* Paratyphi display some similarities to ST313 isolates, in particular some genome degradation (80). Compared with non-iNTS *S*. Typhimurium isolates, ST313 isolates present numerous pseudogenes and deletions (77). In addition, whole-genome comparisons of a representative isolate of ST313, D23580 from Malawi, and ST19 strains (LT2, SL1344, and DT104) revealed a distinct repertoire of six prophage-like elements. These include five full-length prophages arbitrarily named Blantyre Prophage "BTP" 1 through 5, and one prophage remnant (77). Okoro et al. highlighted that this set of prophage sequences is present in all ST313 isolates belonging to lineage I and II (79). Among the five full-length prophages, three of them were already well-characterized, and commonly found in *S*. Typhimurium genomes: Gifsy-2D23580 (BTP2), ST64BD23580 (BTP3), and Gifsy-1D23580 (BTP4) that are all defective in ST313. The two remaining, BTP1 and BTP5, are novel prophages that are found only in the ST313 genome. BTP1 contains three virulencerelated genes: *st313-td*, *gtrCc*, and *gtrAc* (81). *st313-td* was reported to play a role in survival within murine macrophages and in virulence in a mouse model of bacterial infection (82). The *gtrAC* operon encodes an *O*-glycosyltransferase that modifies the composition and the length of O-antigen of the bacterial lipopolysaccharide (83). Further, the LPS of D23580 (ST313) present particular O-polysaccharide chains (84), which have been used to design glycoconjugate vaccines against invasive African *S. enterica* serovar Typhimurium (85, 86).

The D23580 isolate has four plasmids including one virulenceassociated plasmid. This plasmid contains an insertion that resembles a composite *Tn21*-like mobile element encoding multiple drug resistance genes (77). Before the appearance of MDR *S*. Typhimurium, ST313 strains of lineage I were susceptible to chloramphenicol. The selection of this virulence-associated plasmid explains the emergence of MDR *S*. Typhimurium, such as D23580 (lineage II), associated with the epidemic increase in the incidence of iNTS in Malawi after chloramphenicol treatment (74).

After an analysis of 129 ST313 isolates, Yang et al. demonstrated that these exhibit a distinct metabolic signature compared with non-ST313 *S*. Typhimurium. For instance, D23580 seems to be more resistant to acid stress than non-ST313 *S*. Typhimurium (87). Among the differences in metabolic pathways, ST313 strains present two loss-of-function mutations that impair multicellular stress resistance associated with survival outside the host. Hence, ST313 bacteria are less resistant to oxidative stress than ST19 (87, 88), due to mutations causing inactivation of KatE catalase in ST313. Catalase converts hydrogen peroxide (H2O2) to oxygen and water and this H2O2 detoxification protects highdensity bacterial communities from oxidative stress. Another loss-of-function mutation in the *bcsG* gene in D23580 induces an inactivation of the BcsG cellulose biosynthetic enzyme required for the RDAR (red, dry, and rough) colonial phenotype (88). RDAR colonies represent a form of multicellular behavior that enhances *Salmonella* stress resistance in the environment and allows biofilm formation (89). A comparative analysis of biofilmforming ability and long-term survival has shown that ST19 strains, that are strong biofilm producers, can survive desiccation better than ST313 that form weak biofilms and survive poorly following desiccation (90). In addition, several ST313 strains express less flagellin, a component of the flagellum appendage responsible for bacterial motility (91, 92). Indeed, ST313 strains (D65, Q55, S11, S12, D23580, and A130) were reported to be less motile than ST19 strains (I77, I89, I41, S52, and SL1344) (91, 93), although some variability among the strains was observed (87). These data suggest that, like *Salmonella* Typhi, *Salmonella* Typhimurium ST313 lack some of the mechanisms that allow transmission and/or survival in the environment.

### *Salmonella* TYPHIMURIUM AND HOST INTERACTIONS

*Salmonella* Typhimurium were reported to be quickly taken up by CD18<sup>+</sup> cells in the blood after oral infection (94). *Salmonella* Typhimurium are also taken up by CCR6<sup>+</sup> phagocytes located in the subepithelium dome of Peyer's patches in the intestine, then carried to the mesenteric lymph node (95). In addition, dendritic cells were shown to be able to extend dendrites toward the intestinal lumen, without perturbing the tight junctions of epithelial cells, to capture bacteria (96). *Salmonella* Typhimurium persist within macrophages in the mesenteric lymph nodes of chronically infected Nramp1 (natural resistance associated macrophage protein 1)<sup>+</sup>/<sup>+</sup> mice (97). *S.* Typhimurium may be killed by the cell (98) or can hijack the host cell defenses to survive inside the cell (99–101). More recently, it has been recognized that macrophage polarization can influence bacterial infection. A macrophage population is heterogeneous in its susceptibility to the infection, potentially due to a mixture of type 1 and type 2 macrophages, as shown *in vitro* with mouse bone marrow-derived macrophages (102). *S.* Typhimurium cannot replicate in primary human monocyte-derived macrophages polarized into inflammatory M1 macrophages, while M2 and M0 macrophages allowed bacterial replication (103). The tissue source of macrophages further determines the degree of growth or survival of bacteria. For example, *S.* Typhimurium seems to survive better in splenic macrophages than in peritoneal macrophages (104). This is consistent with the main reported site of *Salmonella* infection (105).