Two different but likely complementary mechanisms have emerged from the latest set of studies addressing this subject. On the one hand, Cambier et al. postulated that DIM mask physically mycobacterial PAMPs, and thus prevent their recognition by tolllike receptors (TLR) and the subsequent recruitment of microbicidal macrophages (Cambier et al., 2014). Using zebrafish to model *in vivo* the earliest interactions between bacteria and host macrophages, these authors reported that a *M. marinum* mutant lacking DIM on its surface was unable to prevent the recruitment of iNOS-expressing macrophages, which were effective in killing this mutant through RNS production. Further analysis indicated that DIM act by preventing TLR signaling via the common TLR adaptor MyD88 (Cambier et al., 2014). Supporting this notion, Rousseau et al. reported that infection of murine macrophages with a DIM-deficient *Mtb* mutant induced a high secretion of TNF-α and IL-6, two pro-inflammatory cytokines produced downstream of TLR- and MyD88-dependent signaling pathway (Rousseau et al., 2004). The authors also mentioned that the greater amount of TNF-α and IL-6 at the site of infection could overstimulate the recruitment of macrophages. The exact mechanism of how DIM are able to mask the mycobacterial PAMPs, and which TLR-containing immune cell is involved, remain to be described. On the other hand, our group proposed that DIM target lipid organization in the membrane of host macrophages, thereby modifying its biophysical properties and subsequently the activity of membrane effectors (Astarie-Dequeker et al., 2009). We observed that DIM-deficient mutants are poorly efficient to infect human macrophages, and accumulate in acidified phagosomes at early time post-infection. Importantly, prevention of phagosomal acidification by the proton-ATPase inhibitor, bafilomycine, rescued the growth defect of a DIM deficient mutant (Passemar et al., 2014). This suggests that DIM contribute to the intracellular growth of *Mtb* by excluding somehow the proton-ATPase from the phagosomal membrane. At late post-infection stages, DIM-deficiency affects the capacity of *Mtb* to induce cell death and to disseminate into bystander macrophages (Passemar et al., 2014). While these results should be interpreted with caution, they suggest that DIM participate in control of the outcome of infected macrophages and cell-tocell spread of *Mtb*. At the root of these observations, we noticed that *Mtb* interacts with macrophages in a manner that induces changes in membrane fluidity along with the requirement of DIM at the bacterial surface (Astarie-Dequeker et al., 2009). The ability of DIM to induce changes in membrane fluidity is consistent with their hydrophobic properties that would facilitate their insertion into membranes. As non-covalently bound molecules to the outer cell wall layer of mycobacteria, DIM could thus insert into membranes of macrophages to induce biophysical changes that would modulate the activity of membrane-associated effectors. At the level of the plasma membrane, this would increase macrophage infection by enhancing the activity of phagocytic receptors (e.g., complement receptor 3, CR3), and subsequent bacterial replication through inhibition of phagosome acidification (Astarie-Dequeker et al., 2009; Passemar et al., 2014). Moreover, DIM could also collaborate with the secreted *Mtb* protein, ESAT-6 by modulating its membrane lytic activity. Indeed, DIM and ESAT-6 share the capacity to induce cell death and cellto-cell spread of *Mtb* (Aguilo et al., 2013; Passemar et al., 2014), two events associated to membrane damages.

Collectively, these results clearly established that DIM play an active role at modulating the recognition of mycobacterial PAMPs and avoiding the activation of pro-inflammatory macrophages, and at the same time, altering macrophage responses to its favor possibly by provoking changes in the membrane fluidity that ultimately affect the microbicidal functions.

### **SEEKING THE RIGHT PARTNER TO PLAY: THE SIGNIFICANCE OF PGL STRUCTURAL VARIABILITY**

PGL possess a common lipid core closely related to DIM that, consequently, may share the properties just described for these molecules. However, PGL also exhibit species-specific saccharide domains (**Figure 1**) and this variation in sugar content may have a strong impact on the PRRs involved in their recognition, and therefore, on their biological activities.

Most of *Mtb* clinical isolates, as well as the reference laboratory strains, are unable to produce PGL (Constant et al., 2002). Yet, some *Mtb* strains are able to synthesize a specific form of PGL named PGL-Tb1. This strongly suggests that PGL are not essential for TB pathogenesis. However, their presence may have an impact on the interaction with the host and increases *Mtb* virulence. Indeed, Reed et al. established that the production of PGL-Tb1 in a Beijing clinical isolate is associated with a hypervirulent phenotype in animal models (Reed et al., 2004). It was also reported that treatment of mouse macrophages with purified PGL-Tb1 inhibits the production of the pro-inflammatory cytokines TNF-α, IL-6 and CCL2, and that this effect was dependent on its saccharide domain. The activity of PGL-Tb1 on the modulation of the immune response was also supported by independent results showing that the production of this lipid in the laboratory strain H37Rv, usually unable to synthesize it, modifies cytokine secretion by infected macrophages (Sinsimer et al., 2008). However, in that series of experiments, PGL-Tb1-producing H37Rv was not found to be more virulent than the parental strain in mice. Pointing to the relevance of this molecule, PGL-deficient *Mtb* strains were found to release *p*-hydroxybenzoic acid derivatives (*p*-HBAD), a truncated form of PGL containing just the saccharide domain and the phenol ring, which may also have biological activities (Constant et al., 2002). Mutants unable to produce PGL-Tb1 glycosylated-phenol moiety (*p*-HBAD II) induced increased secretion of the pro-inflammatory cytokines TNF-α, IL-6 and IL-12p40 (Stadthagen et al., 2006).

PGL are also produced by the fish pathogen *M. marinum,* used in several laboratories to model host-pathogen interactions in the context of human TB. PGL-*mar* (**Figure 1**) was found to be a key factor in the phagosomal maturation arrest induced by *M. marinum* (Robinson et al., 2008). In addition, the ability of purified PGL-*mar* to abrogate the secretion of pro-inflammatory cytokines TNF-α and IL-12p40 by human macrophages was also described. Little more was known about the biological activities of this molecule until recently, when it was demonstrated that PGL-*mar* is involved in the chemokine (C-C motif) receptor 2 (CCR2)-dependent recruitment of macrophages by inducing the expression of the chemokine CCL2 through a molecular mechanism still unknown (Cambier et al., 2014). Indeed, by selectively recruiting pathogen-permissive macrophages, Cambier et al. demonstrated that PGL-*mar*, in a proposed coordinated manner with the masking effect of DIM, increases *M. marinum* fitness in the host.

Finally, PGL-1 from *M. leprae* has retained special attention among the different PGL species since its discovery in the early 1980s, mainly due to the high immunogenicity of its sugar moiety (for a review see Spencer and Brennan, 2011). Like PGL-Tb1, PGL-1 contains a trisaccharide domain but the structure is different (**Figure 1**). Mainly due to the inability to grow the leprosy bacillus *in vitro*, there is a large body of literature using isolated PGL-1 to study its role in the modulation of host immune (Mehra et al., 1984; Schlesinger et al., 1994). Among others, PGL-1 was found to inhibit the pro-inflammatory cytokine secretion by human monocytes (Silva et al., 1993). In addition, *M. leprae* induces a poor activation and maturation of dendritic cells, and dampens their ability to induce T-cell responses (Hashimoto et al., 2002). This inhibition was partially relieved by treatment of *M. leprae*-infected cells with anti-PGL-1 antibodies. Together, those studies suggest that PGL-1, through its terminal trisaccharide motif, is the factor responsible for the immunosuppression observed in lepromatous leprosy, one of the most characteristic forms of the disease. Using an original genetic engineering strategy, our team has demonstrated that PGL-1 expression in *M. bovis* BCG enables this recombinant strain to exploit CR3 to promote bacterial uptake and to inhibit TNF-α secretion by human macrophages (Tabouret et al., 2010). In line with this finding, a recent study showed that synthetic PGL-1 saccharide moiety reduces the production of cytokines (TNF-α, IL-6, IL-1β and CCL2) induced by TLR-2 (Elsaidi et al., 2013).

These results indicate that mycobacteria make use of PGL to modulate host innate immune response, and that this activity is primarily mediated by their saccharide domain through an unknown molecular mechanism. Our data also support the notion that some PGL forms, such as PGL-1, could enable mycobacteria to take advantage of CR3 (Tabouret et al., 2010) to invade macrophages and exert anti-inflammatory properties by interfering with the TLR-dependent pro-inflammatory responses, as previously described for other pathogens (Wang et al., 2007; Hajishengallis and Lambris, 2011; Dai et al., 2013). However, we cannot rule out the possibility of a direct inhibition of TLRdependent cytokine secretion, or the direct interaction with a "PGL receptor" on epithelial cells, is involved in the induction of CCL2 and subsequent macrophage recruitment, as hypothesized by Cambier et al. for *M. marinum* (Cambier et al., 2014). Finally, it is important to keep in mind that the structure of the saccharide domain of PGL is highly variable and species-specific (**Figure 1**). Therefore, it remains to be seen whether the strategy involved in immunomodulation is the same or varies for each PGL variant, and whether a PGL variant is more active than others.

### **CONCLUSION: PLAYING HIDE-AND-SEEK WITH HOST MACROPHAGES**

From these data, it emerges that pathogenic mycobacteria utilize DIM, on their own or in conjunction with PGL (depending on mycobacterial strains) for diverting macrophages from their natural function and for establishing infection. Most of the research has focused on the macrophage because its dialog with mycobacteria is thought to be the seminal step of the immune response. The study of Cambier et al. marks an important step in understanding the functions of lipids, as it takes into account the full repertoire of the innate immune system (Cambier et al., 2014). However, several questions remain pending: for example, what is the population of recruited macrophages? If we refer to the model illustrating changes in macrophage polarization throughout the infectious process (Lugo-Villarino et al., 2011), lipids might mediate evasion of microbicidal functions of M1 macrophages in the early stage of infection, whereas they might favor recruitment of M2 macrophages with weak microbicidal competences later on during infection. It also remains to be determined how lipids behave within macrophages with distinct phenotype/polarization in order to assess whether, by virtue of their pleiotropic effect, they have an intracellular impact throughout the disease.

We anticipate that the identification of the molecular mechanisms involved in *Mtb* lipid effects will lead to the characterization of signaling pathways that are modulated for the benefit or detriment of mycobacteria survival in the host. Therefore, we would like to provide a framework to study the molecular mechanisms governing mycobacterial DIM and PGL activity at the direct interface with macrophages (**Figure 2**). We propose that DIM exert more than a masking effect of cell wall exposed *Mtb* PAMPs (Cambier et al., 2014) and play an active role through their hydrophobic part to reshape macrophage activity (**Figure 2A**). We also propose that PGL, on their part, act through their saccharide domains as potential ligands for carbohydrate-recognizing receptors expressed at the surface of macrophages (**Figure 2B**).

For the purpose of conciseness, we focused the current perspective on DIM and PGL, but we believe that in the near future there will be a need for a broad assessment of mycobacterial lipids and their overall effects in the immune system. Novel strategies need to be devised in order to deal with the redundant role as

virulence or immunomodulatory factor among mycobacteria cell envelope lipids. In this aspect, our group has undertaken the construction of single and multiple lipid mutants to assess the role of trehalose-derived lipids, sulfolipids, diacyltrehaloses and polyacyltrehaloses, and their respective contribution to the virulence together with DIM (Passemar et al., 2014). Similarly, there is a well-known redundancy among host PRRs that may hamper the characterization of signaling pathways triggered by mycobacterial lipids; our group has also made headways to develop techniques for the simultaneous double-gene silencing in primary mononuclear phagocytes (Troegeler et al., 2014). Finally, PGL variants merit careful consideration, as there is a strong potential for

bactericidal functions. Likewise, DIM could insert within intracellular membranes (e.g., phagosomal membranes) where they collaborate with

> variation at the functional level according to species. This is particularly true for the assessment of permissive macrophage recruitment toward the site of infection for both PGL-Tb1 and PGL-1. Unlike the inducing effect by PGL-*mar* in the context of zebrafish infection (Cambier et al., 2014), both PGL-Tb1 and PGL-1 inhibit the CCL2 secretion by macrophages (Reed et al., 2004; Elsaidi et al., 2013). This divergence could be due to a differential effect of PGL depending on cellular context, or more interestingly, through a distinctive recognition by the involved receptor according to the saccharide composition. To address this type of question, the strategy developed by our team based on the use of *M. bovis* BCG as surrogate might be a valuable tool to

surface of epithelial cells has been proposed to be responsible for the induction of CCL2, and the subsequent macrophage recruitment.

enable the direct comparison of the effects of the PGL variants in the context of a relevant mycobacterial envelope and within the same genetic background (Tabouret et al., 2010).

In conclusion, we believe that the development of microbiological tools and adequate research models, in combination with multidisciplinary strategies, should open up new venues to achieve a better understanding of the ever-evolving relationship between host and pathogen. In many ways, the lipid at the mycobacteria wall is just a starting point!

### **ACKNOWLEDGMENTS**

The authors received no specific funding for this work. The Guilhot's laboratory is supported by the CNRS, Agence Nationale de la Recherche (ANR) and the Marie Skłodowska-Curie actions fellowship. The Neyrolles's laboratory is supported by CNRS, the European Union (7th framework Programme and ERA-NET), ANR, Fondation pour la Recherche Médicale (FRM), and Institut Mérieux. Both laboratories are supported by the Bettencourt-Schueller Foundation.

### **REFERENCES**

and adjuvanticity of the mycobacterial cord factor and its synthetic analog trehalose-dibehenate. *J. Immunol.* 184, 2756–2760. doi: 10.4049/jimmunol.0904013

**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.

*Received: 23 October 2014; paper pending published: 06 November 2014; accepted: 24 November 2014; published online: 09 December 2014.*

*Citation: Arbues A, Lugo-Villarino G, Neyrolles O, Guilhot C and Astarie-Dequeker C (2014) Playing hide-and-seek with host macrophages through the use of mycobacterial cell envelope phthiocerol dimycocerosates and phenolic glycolipids. Front. Cell. Infect. Microbiol. 4:173. doi: 10.3389/fcimb.2014.00173*

*This article was submitted to the journal Frontiers in Cellular and Infection Microbiology.*

*Copyright © 2014 Arbues, Lugo-Villarino, Neyrolles, Guilhot and Astarie-Dequeker. 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.*

# Manipulation of the endocytic pathway and phagocyte functions by *Mycobacterium tuberculosis* lipoarabinomannan

# *Isabelle Vergne1,2, Martine Gilleron1,2 and Jérôme Nigou1,2\**

*1 Institut de Pharmacologie et de Biologie Structurale, Centre National de la Recherche Scientifique, Toulouse, France 2 Institut de Pharmacologie et de Biologie Structurale, Université Toulouse III - Paul Sabatier, Toulouse, France*

### *Edited by:*

*Soubeyran Philippe, Institut National de la Santé et de la Recherche Médicale, France*

### *Reviewed by:*

*Eric Ghigo, Centre National de la Recherche Scientifique, France Iain Sutcliffe, Northumbria University, UK*

### *\*Correspondence:*

*Jérôme Nigou, Institut de Pharmacologie et de Biologie Structurale, UMR 5089 CNRS - Université de Toulouse, 205 route de Narbonne BP64182, 31077 Toulouse, France e-mail<EMAIL_ADDRESS>
Lipoarabinomannan is a major immunomodulatory lipoglycan found in the cell envelope of *Mycobacterium tuberculosis* and related human pathogens. It reproduces several salient properties of *M. tuberculosis* in phagocytic cells, including inhibition of pro-inflammatory cytokine production, inhibition of phagolysosome biogenesis, and inhibition of apoptosis as well as autophagy. In this review, we present our current knowledge on lipoarabinomannan structure and ability to manipulate the endocytic pathway as well as phagocyte functions. A special focus is put on the molecular mechanisms employed and the signaling pathways hijacked. Available information is discussed in the context of *M. tuberculosis* pathogenesis.

**Keywords: lipoarabinomannan,** *Mycobacterium***, cytokine, phagosome, apoptosis, autophagy**

# **INTRODUCTION**

*Mycobacterium tuberculosis* (*M.tb*), the causative agent of tuberculosis, is one the most effective human pathogens. Its virulence is multifactorial but initially relies on its ability to parasite and manipulate phagocytic cells in the lung.  Indeed, it reproduces several salient properties of *M.tb* in phagocytic cells, including inhibition of proinflammatory cytokines production, inhibition of phagosome maturation, inhibition of macrophage apoptosis, and inhibition of autophagy. ManLAM is a Pathogen-Associated Molecular Pattern recognized by several receptors of the innate immune system, including the C-type lectins Mannose Receptor (MR), DC-SIGN and Dectin-2, as well as TLR2 (Gilleron et al., 2008; Ray et al., 2013). It is a potential ligand for the entry of *M.tb* into macrophages *via* the MR (Schlesinger et al., 1994) and into dendritic cells (DCs) *via* DC-SIGN (Maeda et al., 2003; Tailleux et al., 2003). ManLAM inhibitory properties mainly rely on its ability to bind these two lectins. ManLAM can be a ligand of these receptors not only at the surface of *M.tb* bacilli but also as a soluble molecule. Indeed, it is delivered from infected macrophages, through exosomes or apoptotic vesicles, to noninfected bystander phagocytic cells (Beatty et al., 2000; Schaible et al., 2003). This pathway is thought to be critical for shaping immune response but might also be used by the pathogen as a way to disseminate immunomodulatory molecules such as ManLAM.

### **LIPOARABINOMANNAN STRUCTURE AND PHYSIOLOGICAL ROLE**

Lipoarabinomannan (LAM) is ubiquitously found in mycobacterial species (Nigou et al., 2003; Briken et al., 2004; Gilleron et al., 2008; Mishra et al., 2011; Angala et al., 2014). It presents a tripartite structure including a lipid anchor, namely Mannosyl-Phosphatidyl-*myo*-Inositol (MPI), a polysaccharide backbone composed of D-Mannan and D-Arabinan, and finally caps (**Figure 1A**). MPI anchor is based on a *sn*-glycerol-3-phospho- (1-D-*myo*-inositol) unit with one α-D-Mannopyranosyl (α-D-Man*p*) unit linked at *O*-2 of the *myo*-inositol. Four potential sites of acylation are present on the anchor: positions 1 and 2 of the glycerol unit, position 6 of the Man*p* unit and position 3 of the *myo*-inositol (Nigou et al., 1999; Gilleron et al., 2000) (**Figure 1A**). LAM and its biosynthetic precursors, phosphatidyl*myo*-inositol-mannosides (PIMs) and lipomannan (LM), are predominantly tri- and tetra-acylated by palmitic and tuberculostearic (10-methyl-octadecanoic) acids (Khoo et al., 1995; Gilleron et al., 1999). Position *O*-6 of *myo*-inositol is glycosylated by the mannan core. PIMs comprise different glyco-forms, containing one to six α-D-Man*p* units (PIM1 to PIM6), PIM2 and PIM6 being the most abundant ones. The D-mannan core of LAM and LM is composed of an (α1→6)-Man*p* backbone substituted at some *O*-2 by a single α-D-Man*p* unit. The D-arabinan portion of LAM contains about 60 arabinofuranosyl (Ara*f*) units which are present as a single arabinan chain attached through an (α1→2) linkage near the middle of the D-mannan core (Kaur et al., 2014). The innermost region is made of a linear (α1→5)- Ara*f* backbone and is followed by a branched region. The nonreducing termini consist of branched hexa-arabinofuranosides

**FIGURE 1 | Structural model of and cell signaling pathways triggered by ManLAM and its biosynthetic precursors LM and PIMs. (A)** ManLAM is a 17 kDa heterogenous macromolecule exhibiting a tripartite structure: (i) a MPI anchor, which can be mono- to tetra-acylated, (ii) a polysaccharide backbone composed of D-Mannan and D-Arabinan, and (iii) mannose caps, which are mono-, (α1→2)-di- and (α1→2)-tri-mannoside units. PIMs and LM are biosynthetic precursors of LAM. Their structure is based on the MPI anchor, glycosylated by one to six mannose units (PIMs) or the full mannan domain (LM). To our present knowledge, the MPI anchor and the Mannose caps are the main structural determinants of ManLAM biological properties. **(B)** LM, and to a much lesser extent PIMs, PILAM and ManLAM, induce pro-inflammatory cytokines production in DCs and macrophages *via* the recognition of tri- or tetra-acylated MPI anchor by TLR2/TLR1 heterodimer. ManLAM elicits cytokines in bone marrow-derived DCs *via* mannose caps binding to Dectin-2. But it also inhibits the production of pro-inflammatory cytokines IL-12, TNF-α, and IL-6, and induces IL-10 by LPS-stimulated human DCs through DC-SIGN ligation. The signaling

pathway involves activation of Raf-1, which results in the phosphorylation of the p65 subunit of NF-κB at Ser276, leading to the acetylation of p65 by two histone acetyltransferases. Translocation of NF-κB in response to TLR activation, and initially dedicated to the transcription of the pro-inflammatory cytokine-coding genes, is reoriented on anti-inflammatory promoter targets, resulting in the decrease of these cytokines to the benefit of IL-10. ManLAM also inhibits IL-12 and TNF-α in macrophages independently of IL-10 production, by directly acting on the TLR4 signaling cascade through induced expression of IRAK-M, which can compete with IRAK1 for binding to TRAF6 and thus inhibit NF-κB activation. ManLAM also promotes tyrosine dephosphorylation of multiple proteins including MAPK, an effect that might be explained by an increased activity of tyrosine phosphatase SHP-1. MR is likely to mediate ManLAM immunosuppressive activities in macrophages, although it has no signaling motif in its cytoplasmic domain, raising the intriguing question as to whether it associates with adapter molecules to transduce signals. The ability of ManLAM to bind MR might in part determine its other inhibitory properties as detailed in **Figure 2**.

and linear tetra-arabinofuranosides, which end with an Ara*f*- (β1→2)-Ara*f*-(α1→ motif. Some β-Ara*f* units are substituted at *O*-5 by capping motifs. The caps differ according to the mycobacterial species. LAM from slow-growing mycobacteria, including the pathogenic species *M.tb*, *Mycobacterium leprae* and *Mycobacterium ulcerans*, are capped with mono-, (α1→2)-di- and (α1→2)-tri-mannoside units (mannose-capped LAM is referred to as ManLAM) (Chatterjee et al., 1992) (**Figure 1A**). In contrast, LAM from fast-growing species is either capped by phospho-*myo*inositol units (PILAM), such as in the non-pathogenic model organism *Mycobacterium smegmatis*, or not capped (AraLAM). LAM in any strain further displays considerable structural microheterogeneity, with various acyl-forms and glyco-forms. In addition, ManLAM may be substituted by discrete motifs, such as succinyl residues on the arabinan chain or (α1→4)-linked methyl-thio-D-xylose (MTX) residues on some terminal Man*p* units of the mannose caps or the mannan core.

LAM is not restricted to mycobacteria. Indeed, LAM-like molecules are also produced by phylogenetically close relatives of bacteria of the suborders *Corynebacterineae* and *Pseudonocardineae*, including *Corynebacterium* (Tatituri et al., 2007), *Rhodococcus* (Garton et al., 2002; Gibson et al., 2003b), *Tsukamurella* (Gibson et al., 2004), *Turicella* (Gilleron et al., 2005), *Amycolatopsis* (Gibson et al., 2003a), or *Saccharothrix* (Gibson et al., 2005) genera. In these bacteria, lipoglycans are thought to functionally replace lipoteichoic acid otherwise produced by low G+C Gram-positive bacteria. These macroamphiphiles play a fundamental role in the physiology of bacteria, although yet not fully understood (Ray et al., 2013). Defective or deficient lipoglycans synthesis is associated with lethality or growth defects (Gilleron et al., 2008) and changes in lipoglycan structures have a significant impact on the cell wall integrity of mycobacteria (Fukuda et al., 2013). For example, structural defects in LM and LAM in *M. smegmatis* result in loss of acidfast staining, increased sensitivity to β-lactam antibiotics, and faster killing by macrophages (Fukuda et al., 2013). Accordingly, mycobacterial D-arabinan biosynthesis is the target of ethambutol (Deng et al., 1995), a first-line drug in the treatment of tuberculosis, as well as of benzothiazinones (Makarov et al., 2009), which are new antituberculous drug candidates in preclinical development. The elucidation of the complete biosynthetic pathways of these important molecules is therefore expected to afford novel therapeutic targets (Angala et al., 2014).

In the context of host-pathogen interaction, to our present knowledge, the MPI anchor and the mannose caps are the main structural determinants of ManLAM biological properties, the role of the discrete motifs remaining elusive. The MPI anchor is recognized by TLR2/TLR1 heterodimer, whereas the mannose caps allow the binding to C-type lectins (Gilleron et al., 2008; Ray et al., 2013).

### **MANIPULATION OF PHAGOCYTES RESPONSES**

The success of *M.tb* as an intracellular pathogen relies on its extraordinary capacity to disarm phagocyte antibacterial defenses whereby turning hostile phagocytes into safe havens for replication. Beyond their impact on innate immune responses such manipulations can also be detrimental in development of an efficient adaptive immunity. Interestingly, several of these manipulations can be mirrored by purified ManLAM.

### **PRO-INFLAMMATORY CYTOKINES PRODUCTION**

The inflammatory response is crucial to control *M.tb* infection through macrophage activation and granuloma formation. LM, and to a much lesser extent PIMs, PILAM, and ManLAM, induce pro-inflammatory cytokines production *via* the recognition of tri- or tetra-acylated MPI anchor by TLR2/TLR1 (Gilleron et al., 2003, 2006; Vignal et al., 2003; Quesniaux et al., 2004; Nigou et al., 2008; Ray et al., 2013) (**Figure 1B**). However, a prolonged stimulation of TLR2 has been shown to result in inhibition of MHC class II transactivator expression, MHC class II molecule expression and antigen presentation (Gehring et al., 2004). *M.tb* might have subverted this general mechanism of negative-feedback regulation that prevents excessive T cellmediated inflammation to evade recognition by CD4+ T cells (Harding and Boom, 2010).

ManLAM was recently shown to elicit TNF-α, IL-6, and IL-10 in bone marrow-derived DCs *via* mannose caps binding to Dectin-2 (Yonekawa et al., 2014) (**Figure 1B**). However, we previously found that *M.tb* ManLAM can also inhibit the production of pro-inflammatory cytokines IL-12 and TNF-α by LPS-stimulated human DCs (Nigou et al., 2001, 2002) (**Figure 1B**). We initially proposed the C-type lectin MR to mediate ManLAM inhibitory activity because the latter (i) relied on the presence of both the mannose caps and the fatty acids which are also required for ManLAM binding to MR and (ii) could be mimicked by an agonist anti-MR monoclonal antibody. However, it was later shown that ManLAM binding to DCs is not inhibited by anti-MR but rather by anti-DC-SIGN antibodies and that a blocking anti-DC-SIGN antibody inhibits ManLAM-induced IL-10 production by LPS-stimulated DCs (Geijtenbeek et al., 2003). Why ManLAM only binds DC-SIGN on DCs, although MR is expressed on these cells, remains unclear (Blattes et al., 2013). ManLAM also inhibits IL-12 and TNF-α in human THP-1 (Knutson et al., 1998) and murine RAW 264.7 (Pathak et al., 2005) macrophage cell lines although DC-SIGN is absent. MR is likely to mediate ManLAM effect in these cells, as the ability of MR to trigger an antiinflammatory signal was confirmed by other independent studies (Chieppa et al., 2003; Zhang et al., 2005).

How DC-SIGN or MR signal into the cells and interfere with LPS-induced TLR4 signaling is not yet completely understood (**Figure 1B**). DC-SIGN displays intracellular motifs that are able to constitutively recruit the lymphocyte-specific adaptor protein LSP1 which associates the complex KSR1-CNK-Raf-1 (Gringhuis et al., 2009) (**Figure 1B**). Upon ligand binding, activation of Raf-1 results in the phosphorylation of the p65 subunit of NF-κB at Ser276, leading to the acetylation of p65 (Gringhuis et al., 2007). NF-κB activity is then prolonged and increases the transcription rate at the IL-10 anti-inflammatory cytokine promoter. However, Raf-1 signaling alone does not induce cytokine expression. Translocation of NF-κB in response to TLR activation and initially dedicated to the transcription of the pro-inflammatory IL-12p35, IL-12p40, IL-6, and TNF-α cytokine-coding genes is reoriented on anti-inflammatory promoter targets, resulting in the decrease of these cytokines to the benefit of IL-10 (Gringhuis et al., 2007) (**Figure 1B**). Gringhuis et al. (2009) proposed that DC-SIGN may discriminate among mannosylated and fucosylated ligands and modulate the TLR signaling into a pro- or anti-inflammatory response respectively. However, this appears to be in contradiction with the set of data showing that ManLAM or synthetic mannosylated analogs engaging DC-SIGN inhibit pro-inflammatory cytokines production (Nigou et al., 2001; Geijtenbeek et al., 2003; Blattes et al., 2013). MR has no signaling motif in its cytoplasmic domain, raising the intriguing question as to whether it associates with adapter molecules to transduce signals. Pathak et al. (2005) demonstrated that ManLAM dampens IL-12 in RAW 264.7 macrophages independently of IL-10 production, by directly acting on the TLR4 signaling cascade through induced expression of IRAK-M, which can compete with IRAK1 for binding to TRAF6 and thus inhibit NF-κB activation (**Figure 1B**).

ManLAM anti-inflammatory activity relies on its ability to bind DC-SIGN or MR and both the mannose caps and the fatty acids are required for efficient binding (Nigou et al., 2001, 2002). Indeed, fatty acids induce a supramolecular organization of ManLAM in aqueous solution, resulting in the formation of a 30 nm spherical structure (**Figure 1B**), composed of approximately 450 molecules with the mannose caps exposed at the surface (Riviere et al., 2004). This multivalent supramolecular structure allows multipoint attachment of ManLAM, *via* mannose caps, to the Carbohydrate Recognition Domains (CRD) of multimeric DC-SIGN receptors (Feinberg et al., 2001; Mitchell et al., 2001), thereby ensuring high affinity binding (Nigou et al., 2001; Riviere et al., 2004) (**Figure 1B**). Following this rationale, we were able to design fully synthetic compounds mimicking the bioactive supramolecular structure of ManLAM, i.e., mannodendrimers, that display potent anti-inflammatory activity both *in vitro* and *in vivo* and that could be of therapeutic use (Blattes et al., 2013).

The ability of ManLAM to bind MR might in part determine its other inhibitory properties as described below (**Figure 1B**) (Gilleron et al., 2008).

### **PHAGOSOME MATURATION**

One main function of professional phagocytes is the uptake of microorganisms through phagocytosis. This event results in formation of a vacuole called phagosome which then matures into a phagolysosome through a series of fusion reactions with the endocytic and secretory pathways and ultimately fusion with lysosomes (Flannagan et al., 2012). Maturation endows phagosome with new bactericidal properties predominantly hydrolase activities, acidic pH and antimicrobial peptides. Therefore, phagosome maturation process is crucial for killing of captured microbes as well as their antigen presentation to T lymphocytes. Inhibition of phagosome maturation by *M.tb* was reported more than 40 years ago (Armstrong and Hart, 1971). Since then, numerous mycobacterial factors have been identified and characterized as disruptors of phagosome maturation (Russell, 2011), including ManLAM and PIMs (Fratti et al., 2001; Vergne et al., 2004). Importantly, the ability of *M.tb* to block phagosome maturation is shared by other pathogenic mycobacteria such as *Mycobacterium avium* and *Mycobacterium marinum* which produce ManLAM but not by non-pathogenic *M. smegmatis* which produces PILAM (Anes et al., 2006; Appelmelk et al., 2008; de Chastellier et al., 2009).

Early work by Deretic and colleagues showed that mycobacteria block phagosome maturation between stages orchestrated by Rab5 and Rab7, two small GTPases involved in membrane trafficking and present on early and late endosomes, respectively (Via et al., 1997). Later on, they pinpointed this block to impairment in recruitment of EEA1, a tethering protein and Rab5 effector, essential for phagosome maturation (Fratti et al., 2001). EEA1 recruitment is instrumental in delivering hydrolases such as Cathepsin D and H+-ATPase subunit Vo from Trans-Golgi-Network (TGN) to the phagosome (Fratti et al., 2003). EEA1 is recruited to the phagosomal membrane via Rab5 and phosphatidylinositol 3-phosphatase (PI3P) which is synthesized by type III PI3Kinase, hVPS34 (Fratti et al., 2001). In the same report, authors showed that ManLAM-coated beads, in contrast to control beads, prevent EEA1 recruitment to the phagosomal membrane, delivery of Cathepsin D, and phagosome acidification (Fratti et al., 2001, 2003). Inhibition of phagosome maturation by ManLAM was later confirmed by several groups (Hmama et al., 2004; Kang et al., 2005; Welin et al., 2008).

Another important player in phagosome maturation is Ca2+ signaling. Phagocytosis of dead *M.tb* but not of live *M.tb* triggers an increase of cytosolic Ca2+ that results in activation of calmodulin-dependent kinase II (CaMKII) (Malik et al., 2000, 2001). Inhibition of Ca2+, Calmodulin (CaM) and CaMKII prevents phagosome containing dead *M.tb* to fuse with lysosomes. Vergne et al. showed that Ca2+ signaling is central for PI3P synthesis on phagosomal membrane, consequently for EEA1 recruitment (Vergne et al., 2003). CaM and CaMKII seem to play a role in hVPS34 recruitment and/or activation. Notably, in contrast to PILAM, ManLAM limits Ca2+ influx in cytosol, thus explaining its effect on EEA1 recruitment and phagosome maturation (**Figure 2**). Interestingly, PIMs can also inhibit phagosome acidification, not by preventing EEA1 recruitment, but by promoting fusion between phagosome and early endosomes (Vergne et al., 2004). How PIMs trigger early endosome fusion remains to be elucidated but it might involve Rab14, a small GTPase specifically recruited by live mycobacteria to favor phagosome-early endosomes fusion and block phagosome acidification (Kyei et al., 2006) (**Figure 2**).

What are ManLAM molecular targets, upstream of Ca2+ signaling, responsible for phagosome maturation arrest? Two main mechanisms, non-mutually exclusive, have been uncovered. Schlesinger's group has demonstrated that ManLAM limits phagosome maturation by binding to MR (Kang et al., 2005). Interestingly, ManLAM acyl chains are important to maintain this blockade beyond 1 h, suggesting a possible additional mechanism for ManLAM action. ManLAM can insert into lipid microdomains, called rafts, *via* the MPI anchor, resulting in membrane disorganization and inhibition of membrane fusion (Hayakawa et al., 2007; Welin et al., 2008). However, it is still unclear whether rafts disruption and/or MR are responsible of Ca2+ signaling inhibition or are completely independent mechanisms.

### **APOPTOSIS**

The role(s) of apoptosis and other cell-death pathways in Tuberculosis remain(s) a matter of intense debate. Several reports suggest that inhibition of excessive apoptosis may be beneficial for the pathogen during early stage of infection for maintaining its replicative niche and limiting cross-presentation and crosspriming of CD8+ T-cells through phagocytosis of apoptotic bodies by DCs. *M.tb* seems to be able to block both extrinsic pathway of host cell apoptosis which relies on activation of death receptors (Fas/CD95, TNFR1) and the intrinsic pathway triggered

**FIGURE 2 | Schematic representation of ManLAM and PIM action on phagosome maturation and autophagy. Right part:** After phagocytosis, mycobacteria reside in a vacuole, called phagosome. Phagosome maturation consists in a series of fusion events with exocytic and endocytic pathways. One key step is the delivery of Cathepsin D and H+-ATPase subunit Vo from Trans-Golgi-Network (TGN) to the phagosome. This step is mediated by tethering protein EEA1 which is recruited to the phagosome by small GTPase Rab5 and phosphatidylinositol 3-phosphate (PI3P). ManLAM blocks phagosome maturation through inhibition of Ca2+/CaM/CaMKII signaling pathway involved in PI3P production by type III PI3Kinase hVPS34. ManLAM can also block phagosome maturation by engaging mannose receptor and disrupting membrane microdomains, rafts, however, the link with Ca2+/CaM signaling has not been studied. PIMs, ManLAM precursor, impair phagosome maturation by stimulating fusion between phagosome and early endosomes. *Mycobacterium tuberculosis* recruits Rab14 to phagosome to promote early endosome fusion thus impairs phagosome maturation. It remains to be established whether PIMs promotes early endosome fusion through Rab14 recruitment. **Left part:** Mammalian target of rapamycin (mTOR) kinase, activated by Ser/Thr kinase Akt and inhibited by AMP-activated Protein Kinase (AMPK), is a master repressor of autophagy. Beclin-1, an autophagy-related protein in complex with hVPS34, is essential for autophagy. Beclin-1/hVPS34 complex is activated by AMPK and repressed by Bcl-2. ULK1, another important autophagy-related protein, is activated by AMPK and inhibited by mTOR. Ca2+ influx has been shown to activate AMPK, hVPS34, and represses Bcl-2 expression. IFNγ induces autophagy. Based on known effects of ManLAM on Ca2+ influx, Bcl-2, Akt and IFNγ signaling, we postulate that ManLAM might inhibit autophagy by targeting Beclin-1/hVPS34 complex, Akt/mTOR or IFNγ pathways. The relationship between effects of ManLAM on these different signaling pathways and autophagy awaits investigation. Arrows and characters are represented in gray to indicate that the molecular mechanisms of LAM action on autophagy are hypothetical.

by mitochondrial outer membrane permeabilization (Briken and Miller, 2008). Although *M.tb* genes involved in these inhibitions are just beginning to be unveiled, ManLAM was one of the first mycobacterial product identified as an inhibitor of apoptosis (Rojas et al., 2000; Briken and Miller, 2008).

The mechanisms of apoptosis inhibition by ManLAM seem to be multiple. As for phagosome maturation, ManLAM inhibition of Ca2+ signaling appears to be an important step in blocking infection-induced apoptosis (Rojas et al., 2000). Numerous Ca2+-associated events are known to play a role in apoptosis, among them alteration of mitochondrial permeability transition and down-regulation of anti-apoptotic protein Bcl2 have been shown to be repressed by ManLAM (Rojas et al., 2000). Besides Ca2+ signaling, ManLAM can prevent intrinsic apoptosis pathway through activation of Ser/Thr kinase Akt and phosphorylation of the pro-apoptotic protein Bad (Maiti et al., 2001). More recently, one report indicates that ManLAM can promote extracellular release of soluble TNF-α receptor. Thus, ManLAM might also interfere with the extrinsic apoptosis pathway by neutralizing TNF-α (Richmond et al., 2012).

### **AUTOPHAGY**

Autophagy is a highly conserved eukaryotic intracellular process that carries out lysosomal degradation of damaged, superfluous or toxic cytoplasmic components (Levine et al., 2011; Rubinsztein et al., 2012). In addition to its housekeeping role, autophagy plays major immunological functions, especially, in host antibacterial defenses (Levine et al., 2011; Deretic et al., 2013). These functions range from effector of pattern recognition receptors and inflammation regulation to antigen presentation and direct elimination of microbial agents. Specifically, autophagy is a key immune effector involved in intracellular clearance of important bacterial pathogens such as *M.tb*.

Autophagy is orchestrated by more than 30 dedicated proteins, called autophagy-related proteins (Atg) (Marino et al., 2014). The autophagic process begins with formation of an isolation membrane initiated by Ser/Thr kinase Ulk1 (Atg1), which phosphorylates Beclin-1 (Atg6) in complex with hVPS34 to promote its activation (Russell et al., 2013). The isolation membrane is then expanded through action of two ubiquitinlike conjugation systems, the covalent linkage of Atg12 with Atg5 and of LC3 (Atg8) with phosphatidylethanolamine, which lead to engulfment of intracellular components inside a doublemembrane bound organelle called autophagosome. LC3, along with entrapped cytosolic content, is then degraded after fusion of autophagosome with lysosomes. In the context of phagocytosis, a non-canonical autophagy pathway, called LC3-associated phagocytosis (LAP), has been described which involves direct LC3 lipidation on the phagosomal membrane (Mehta et al., 2014). This alternative pathway, triggered by some Pattern Recognition Receptors, such as TLR2, appears to be ULK1 independent and important in modulating innate immune response (Mehta et al., 2014). However, the detailed molecular mechanisms and the functional role(s) of LAP still remain to be fully elucidated.

*M.tb*, like other intracellular intracellular pathogens, has developed mechanisms to manipulate autophagic pathway (Huang and Brumell, 2014).  Likewise, macrophage treatment with ManLAM for 24 h results in diminution of autophagy as seen by LC3 immunoblotting (personal observation). Autophagy-related proteins play major roles in mediating IFNγ-induced host defenses (Levine et al., 2011; Deretic et al., 2013). Since ManLAM can repress IFNγ responses, it is tempting to speculate that ManLAM might also interfere with autophagy in this context (Sibley et al., 1988; Chan et al., 1991). The action mechanism of ManLAM on autophagy has not been revealed yet, but based on its effect on hVPS34 in phagosome maturation one can postulate that it might inhibit autophagy by modulating hVPS34 in complex with Beclin-1 (**Figure 2**). In addition, Bcl-2 interacts with Beclin-1 to block autophagy, thus ManLAM might impair autophagy via upregulation of Bcl-2 expression (Rojas et al., 2000; Pattingre et al., 2005). ManLAM inhibition of Ca2+ influx could also affect the Ca2+/AMP-activated protein kinase (AMPK) signaling pathway involved in mammalian target of rapamycin (mTOR) kinase- and ULK1-dependent autophagy (Vergne et al., 2003; Alers et al., 2012). Alternatively, ManLAM might repress autophagy through activation of type I PI3Kinase and Akt (Maiti et al., 2001; Ravikumar et al., 2010). Further investigations are definitely required to better understand how ManLAM interferes with autophagy, whether it represses canonical autophagy and/or LAP and what is the significance of this inhibition in terms of phagosome trafficking and phagocyte survival.

### **CONCLUSION**

ManLAM, as a purified molecule, reproduces several salient inhibitory properties of *M.tb* in phagocytic cells. However, the role played by ManLAM in the context on an infection by *M.tb* remains unclear. ManLAM immunosuppressive activities rely on the presence of the mannose caps. But, an *M.tb* mutant lacking the mannose caps on LAM was not affected for its virulence in mice nor for its interaction with phagocytic cells *in vitro* (Appelmelk et al., 2008; Afonso-Barroso et al., 2012). In contrast, an aptamer against ManLAM was found to inhibit *M.tb* infection in mice and Rhesus monkeys (Pan et al., 2014). Moreover, protein LprG, which binds ManLAM and determines its cell surface localization, was found to be essential for virulence of *M.tb* and to control phagolysosomal fusion (Gaur et al., 2014; Shukla et al., 2014). These data are not necessarily contradictory. Indeed, the envelope of mycobacteria is exceptionally rich in mannoconjugates bearing (α1→2)-oligomannosides, including LM, PIM6, arabinomannan or mannoproteins that are able to bind C-type lectins (Pitarque et al., 2005; Torrelles and Schlesinger, 2010). Of note, LprG has been shown to bind LM and PIMs, in addition to ManLAM (Drage et al., 2010), suggesting that the role of LprG might not be attributable to ManLAM inhibitory activities only. Altogether, data converge to indicate that DC-SIGN/MR ligands are most probably redundant at the *M.tb* cell surface, possibly because targeting these receptors is mandatory for the pathogen to manipulate and survive inside the infected host.

### **ACKNOWLEDGMENTS**

The work of our team related to the topic reviewed in this article was supported by CNRS, University of Toulouse, Fondation pour la Recherche Médicale, Invivogen and grants from the Agence Nationale de la Recherche (ANR-05-MIIM-038- 02), the European Union (LSHP-CT-2007-037388; LSHP-CT-2006-037326; HEALTH-F7-2009-241745), the National Institutes of Health (AI064798) and the Indo-French Centre for the Promotion of Advanced Research (IFCPAR/CEFIPRA; 3403-1). Isabelle Vergne is supported by a European Union FP7 Marie Curie Career Integration Grant Europe (autophagtuberculosis).

### **REFERENCES**

Glycoconjugate, Lipoarabinomannan. *J. Biol. Chem.* 289, 30249–30256. doi: 10.1074/jbc.M114.599415

**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.

*Received: 03 November 2014; accepted: 15 December 2014; published online: 12 January 2015.*

*Citation: Vergne I, Gilleron M and Nigou J (2015) Manipulation of the endocytic pathway and phagocyte functions by Mycobacterium tuberculosis lipoarabinomannan. Front. Cell. Infect. Microbiol. 4:187. doi: 10.3389/fcimb.2014.00187*

*This article was submitted to the journal Frontiers in Cellular and Infection Microbiology.*

*Copyright © 2015 Vergne, Gilleron and Nigou. 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.*

# *Coxiella burnetii* lipopolysaccharide blocks p38**α**-MAPK activation through the disruption of TLR-2 and TLR-4 association

#### *Filippo Conti 1†, Nicolas Boucherit 1†, Veronica Baldassarre1, Virginie Trouplin1, Rudolf Toman2, Giovanna Mottola3,4, Jean-Louis Mege1 and Eric Ghigo1 \**

*1 CNRS UMR 7278, IRD198, INSERM U1095, Aix-Marseille Université, Marseille, France*

*2 Laboratory for Diagnosis and Prevention of Rickettsial and Chlamydial Infections, Institute of Virology, Slovak Academy of Sciences, Bratislava, Slovakia*

*3 UMR MD2, Faculté de Médecine NORD, Aix-Marseille Université and IRBA (Institute of Research in Biology of the French Army), Marseille, France*

*4 Laboratory of Biochemistry, La Timone University Hospital, Assistance Publique Hôpitaux de Marseille, Marseille, France*

### *Edited by:*

*Benjamin Coiffard, Hôpital Nord, Marseille, France*

### *Reviewed by:*

*Ravindra Taware, National Chemical Laboratory, India Massimo Mallardo, University of Naples, Italy*

#### *\*Correspondence:*

*Eric Ghigo, Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes, Faculté de Médecine, 27 Bd. Jean Moulin, 13385 Marseille Cedex 05, France e-mail<EMAIL_ADDRESS>
*†These author have contributed equally to this work.*

To survive in macrophages, *Coxiella burnetii* hijacks the activation pathway of macrophages. Recently, we have demonstrated that *C. burnetii*, via its lipopolysaccharide (LPS), avoids the activation of p38α-MAPK through an antagonistic engagement of Toll-like receptor (TLR)-4. We investigated the fine-tuned mechanism leading to the absence of activation of the p38α-MAPK despite TLR-4 engagement. In macrophages challenged with LPS from the avirulent variants of *C. burnetii*, TLR-4 and TLR-2 co-immunoprecipitated. This association was absent in cells challenged by the LPS of pathogenic *C. burnetii*. The disruption makes TLRs unable to signal during the recognition of the LPS of pathogenic *C. burnetii*. The disruption of TLR-2 and TLR-4 was induced by the re-organization of the macrophage cytoskeleton by *C. burnetii* LPS. Interestingly, blocking the actin cytoskeleton re-organization relieved the disruption of the association TLR-2/TLR-4 by pathogenic *C. burnetii* and rescued the p38α-MAPK activation by *C. burnetii*. We elucidated an unexpected mechanism allowing pathogenic *C. burnetii* to avoid macrophage activation by the disruption of the TLR-2 and TLR-4 association.

**Keywords: TLR-2, TLR-4, cytoskeleton,** *Coxiella burnetii,* **macrophages**

# **INTRODUCTION**

*Coxiella burnetii* is an intracellular bacteria responsible of the Q fever zoonosis and is a potential bio warfare and bioterrorism agent (Regis, 1999; Madariaga et al., 2003). Q fever is characterized by a lethal endocarditis (Raoult et al., 2005). It has been shown that molecular variations in *C. burnetii* lipopolysaccharide (LPS) between LPS from virulent and avirulent *C. burnetii* (vLPS and avLPS, respectively) determine the pathogenic properties of *C. burnetii* (Lukacova et al., 2008; Toman et al., 2009; Toman and Vadovic, 2011; Barry et al., 2012 ˇ ).

To survive in macrophages, *C. burnetii* inhibits phagolysosome biogenesis (Ghigo et al., 2002; Barry et al., 2012) and induces cytoskeleton rearrangement of macrophages (Meconi et al., 1998; Honstettre et al., 2004). It has been demonstrated that LPS is the principal actor of the survival mechanism of *C. burnetii* (Meconi et al., 1998; Honstettre et al., 2004; Barry et al., 2012). vLPS stimulates morphologic changes characterized by an intense and transient membrane rearrangement of F-actin leading to protrusions and polarized projections, whereas avLPS does not induce any modification of the cell cytoskeleton morphology (Meconi et al., 1998; Honstettre et al., 2004). In addition, *C. burnetii* targeting to degradative compartments also involves an antagonistic engagement of Toll-like receptor (TLR)-4 by vLPS, lack of p38α-MAPK-driven phosphorylation, and block in recruitment of the HOPS (homotypic fusion and protein-sorting complex) component Vps41 to vLPS-containing vesicles (Barry et al., 2012).

In response to LPS stimulation, TLR-signaling initiates distinct innate immune defensive programs, such as the maturation of phagosomes (Blander and Medzhitov, 2004, 2006). This process involves crosstalk between mitogen-activated protein kinase (MAPK) signaling and components of the vesicular trafficking machinery (Blander and Medzhitov, 2006; Symons et al., 2006; Fontana and Vance, 2011). TLR-4 is involved in the recognition of Gram-negative bacteria such as *E. coli* through recognition of prototypic LPS. TLR-2 interacts with Gram-positive bacteria following interaction with lipoproteins, proteoglycans or lipopeptides. However, several studies have highlighted that LPS recognition is not restricted to TLR-4. Indeed, TLR-2 is able to recognize the LPS from *Porphyromonas gingivalis* (Medzhitov, 2001; Underhill, 2004). Recent studies have highlighted that TLR-2 is required along with TLR-4 for the response to bacterial LPS (Good et al., 2012); this response involves a physical interaction between TLR-2 and TLR-4 (Lee et al., 2004; Good et al., 2012). Much remains to be learned regarding the molecular basis underlying the crosstalk between the LPS variants and TLRs.

In this study, we investigated the mechanism leading to the absence of activation of the p38α-MAPK despite TLR-4 engagement by *C. burnetii* vLPS. We found that the association between TLR-2 and TLR-4 is required to activate p38α-MAPK and was disrupted by the vLPS. The disruption of TLR-2 and TLR-4 association by vLPS was induced by the re-organization of the macrophage cytoskeleton. Interestingly, the block of the actin cytoskeleton re-organization inhibited the disruption of association TLR-2/TLR-4 by pathogenic *C. burnetii* and allowed the p38α-MAPK activation by *C. burnetii* LPS. We elucidated an unexpected mechanism allowing pathogenic *C. burnetii* to avoid activating macrophages.

### **RESULTS**

### **vLPS DISRUPTS TLR-4 AND TLR-2 ASSOCIATION AT THE BMDMs MEMBRANE SURFACE**

In wild-type BMDMs, vLPS was unable to induce the activation of p38α MAPK (*<*0 RFUs), in contrast to the avLPS (13.5 RFUs at 30 min) (**Figure 1**) as previously described (Barry et al., 2012). These data confirm the previous finding that the recognition of vLPS by TLRs is required to block p38α MAPK activation (Barry et al., 2012). We decided to deepen our analysis by investigating the distribution of TLR-4 and TLR-2 at the membrane surface of BMDMs challenged with *C. burnetii* LPSs (**Figure 2**). In control BMDMs, we observed a large number of TLR-2 and TLR-4 fluorescent small dots (129.2 ± 12.3 and 109.5 ± 18 a.u, respectively) with a dispersed distribution at the macrophage surface (**Figures 2A–C**) with a reduced area (5 ± 2 and 9 ± 5 a.u, respectively) (**Figures 2A,B,D**). In macrophages treated with the avLPS, we found a significant decrease in TLR-2 (4.5-fold) and TLR-4 (2.8-fold) dots compared to the control (**Figures 2A–C**) at the macrophage surface. The decreased dot number is associated with an increase in dot area (**Figures 2A,B,D**). Indeed the TLR-2 and TLR-4 dot area increased significantly 7.6-fold and 4.4 fold, respectively, compared to the control (**Figures 2A,B,D**). vLPS induced a significant a decrease of 3.6-fold of the small dots numbers present at cell membrane (**Figures 2A–C**) associated with a significant reorganization of TLR-2 small dots in large patch compared to control (23 ± 3 a.u vs. 5 ± 2) (**Figures 2A,B,D**). Interestingly, vLPS does not affect the distribution or the size of TLR-4 fluorescent dots at the BMDMs membrane surface. Then, we assessed the co-localization of TLR-4 with TLR-2 in BMDMs treated with *C. burnetii* LPSs (**Figures 2E**). In macrophages challenged with avLPS we find a strong co-localization of TLR-4 with TLR-2 (Pearson's coefficient 0.72 ± 0.12) whereas in presence of vLPS we found that TLR2 did not co-localize with TLR-4 (Pearson's coefficient 0.1 ± 0.02). Next, we investigated, by co-immunoprecipitation, the association of TLR-2 with TLR-4 in BMDMs either challenged or not challenged with vLPS or avLPS (**Figure 2F**). We observed that TLR-2 and TLR-4 coimmunopreciptated in the BMDMs control as well in BMDMs challenged with avLPS. In contrast, TLR-2 and TLR-4 did not co-immunoprecipitate in macrophages treated with vLPS. Taken together this data suggests that vLPS disrupts the membrane distribution of TLR-2 and TLR-4.

### **CYTOSKELETON RE-ORGANIZATION INDUCED BY vLPS DISRUPTS P38α MAPK ACTIVATION THROUGH TLRs**

vLPS is known to induce cytoskeleton reorganization (Meconi et al., 1998; Honstettre et al., 2004). We postulated that this

expressed as normalized RFU and represent the mean ± SD (*n* = 3).

reorganization could influence the TLR-2 and TLR-4 distribution observed in BMDMs challenged with vLPS. We have evaluated the capacity of *C. burnetii* LPS to induce cytoskeleton reorganization. We observed, as previously described, that vLPS induced a dramatic reorganization of the BMDMs cytoskeleton (**Figure 3A**), whereas avLPS did not (**Figure 3B**) (Meconi et al., 1998; Honstettre et al., 2004). vLPS induced macrophage spreading and the formation of polarized filopodia and lamellipodia. F-actin was concentrated beneath filopodia and lamellipodia and as spots in cytoplasmic areas (**Figure 3A**). In contrast, avLPS had a slight effect on F-actin organization (**Figure 3B**). After 10 min of stimulation with vLPS, 81 ± 4% of macrophages exhibited filopodia, and the percentage of macrophages with filopodia decreased thereafter (**Figure 3C**). Only 35% of cells treated with avLPS exhibited filopodia (**Figure 3C**). Next, we have investigated if the block of the cytoskeleton re-organization could rescue the association between TLR-4 and TLR-2. We inhibited cytoskeleton re-organization using cytochalasin-D (**Figure 3C**). We revealed that in presence of cytochalasin-D, TLR-2 and TLR-4 co-immunoprecipitate in contrast to the experimental condition without the inhibitor (**Figure 3D**). Finally, we found that the inhibition of the cytoskeleton reorganization by cytochalasin-D recovers activation of p38α MAPK via vLPS (14.1 ± 0.56 RFUs after 90 min), whereas in the absence of cytochalasin-D, p38α MAPK is not activated (**Figure 3E**).

### **DISCUSSION**

*C. burnetii*, the bacteria that causes Q fever, has evolved several strategies to survive in macrophages. One of these strategies is to avoid being targeted to the degradative compartments of immune cells. To do that *C. burnetii*, through its vLPS, blurs its own recognition by TLR receptors in order to interfere with the transduction of the signal (Barry et al., 2012). The consequences of such strategies are a deficiency of p38α-MAPK-driven phosphorylation and a block in recruitment of the homotypic fusion. In addition, several years ago it was shown that *C. burnetii* interferes with the cytoskeleton, and this inference is crucial

**FIGURE 2 | TLR-2 and TLR-4 distribution and colocalization.** BMDMs from wild type mice were challenged for 5 min with *C. burnetii* LPS (1μg/ml). The distribution of **(A)** TLR-2 and **(B)** TLR-4 at the BMDMs surface was determined by confocal microscopy. The scale bar indicates 5μm. The number of TLRs signal detected **(C)** and the area **(D)** were quantified using ImageJ software. The results are expressed as the mean ± SD (*n* = 3, ∗*p <* 0*.*05). **(E)** The colocalization of TLR-2 with TLR-4 was determined using

confocal microscopy. The colocalization of TLR-2 with TLR-4 was quantified using ImageJ software. The results are expressed as the mean ± SD (*n* = 3, ∗*p <* 0*.*05). The scale bar indicates 5μm. **(F)** BMDMs in non-starved conditions were either left untreated or treated with vLPS or avLPS (1 μg/ml) for 5 min, then TLR-2 was immunoprecipitated and coimmunoprecipitated with TLR-4 was visualized by immunoblotting. The blot shown is representative of three experiments.

immunoprecipitated and coimmunoprecipitated with TLR-4 was visualized by immunoblotting. The blot shown is representative of three experiments. **(E)** BMDMs from wild type mice were challenged with vLPS (1 μg/ml) for different periods (min) in presence or not of cytochalasin-D, and the phosphorylation of p38α MAPK was determined using phospho-p38α MAPK cell-based ELISA. The results are expressed as normalized RFU and represent the mean ± SD (*n* = 3).

for its survival in macrophages (Meconi et al., 1998; Honstettre et al., 2004). We have investigated if the dramatic cystoskeleton re-organization induced by *C. burnetii* could explain the decrease of p38a MAPK signaling. In macrophages challenged with *C. burnetii* avLPS we observed a phosphorylation of the p38α MAPK. In contrast, p38α MAPK is not activated in macrophages challenged with vLPS has previously described (Barry et al., 2012). Because it is known that TLR-2 is required along with TLR-4 for the response to bacterial LPS (Good et al., 2012), through a physical interaction between TLR-2 and TLR-4 (Lee et al., 2004; Good et al., 2012) we have analyzed the distribution of TLR-2 and TLR-4 at the surface of macrophages via confocal microscopy. We have observed that C*. burnetii* vLPS induces a strong reorganization of the TLR-2 and TLR4 at the membrane. This redistribution hampers the colocalization between TLR-2 and TLR-4, in contrast to what is observed in macrophages challenged by avLPS. In addition, the co-immunopreciptation experiments highlight a physical link between TLRs in cells challenged with avLPS, whereas in cells treated with vLPS this is not found. Finally, we postulated that the TLR distribution was linked to the cytoskeleton re-organization induced by *C. burnetii* vLPS. Interestingly, the inhibition of the cytoskeleton reorganization by cytochalasin D allowed for the activation of p38α MAPK by vLPS. We observed also that in this condition TLR-4 co-immunoprecipitated with TLR-2. A possible mechanism to explain the role of the vLPS in the default of activation of p38α MAPK is that vLPS through the induction of the cytoskeleton remodeling, induces a relative dispersion and redistribution of TLR-2, TLR-4 receptors at outer membrane level, in such a way that TLR-2 and TLR-4 are not able to signal via p38a MAPK. Moreover, it has been already reported that a crosstalk between TLRs signaling and G-protein coupled receptors, such as Rho-GTPase and Rnd proteins, exists and could lead to a dramatic cytoskeleton rearrangement (Ruse and Knaus, 2006). Previously, we have demonstrated that *C. burnetii* vLPS interferes with phagosome maturation by inhibiting the activation of p38α MAPK (Barry et al., 2012), here we deepened the previous study by demonstrating that *C. burnetii*, through it vLPS blurs the TLR-2 and TLR-4 signaling through dramatic cytoskeleton reorganization and redistribution of TLR-2 and TLR-4 at the macrophage cells surface.

### **MATERIALS AND METHODS ETHICS STATEMENT**

All animal experiments were conducted according to the Guiding Principles of Animal Care and Use defined by the Ethics Committee for Animal Experimentation (N◦14 designated by the National Study Committee on the Ethics of Animal Experimentation) according to the rules of Decree N◦87-848 as of October 19, 1987. All of the animal experiments conducted in this study were also approved by the Ethics Committee for Animal Experimentation (N◦14 from the National Study Committee on the Ethics of Animal Experimentation) where the experiments were performed (Faculty of Medicine, Marseille, experimentation permit number to Eric Ghigo 10-300122013).

### **ANTIBODIES AND FLUORESCENT COMPOUNDS**

**A**ntibodies specific for TLR-2 and TLR-4 were purchased from the BD Bioscience. Cytochalasin D was purchased from Sigma-Aldrich. Secondary antibodies and phalloidin alexa-448 were purchased from Invitrogen

### **LPS PREPARATIONS**

LPS from virulent (vLPS) and avirulent (avLPS) *C. burnetii* (Barry et al., 2012) were isolated from *C. burnetii* RSA 493 (clone 7) and RSA 439 (clone 4), as previously described (Skultety et al., 1996; Toman and Skultety, 1996). The quality of the LPS preparation was confirmed using silver staining and compositional GC-MS (Toman et al., 2009).

### **CELL CULTURE**

Bone marrow-derived macrophages (BMDMs) were generated from 6- to 8-week-old C57BL/6 mice, as previously described (Ren et al., 2005; Cook et al., 2007; Trouplin et al., 2013). BMDM were grown in DMEM supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 IU/ml penicillin, and 100μg/ml streptomycin at 37◦C in 5% CO2. For some experiments macrophages were challenged with 1μg/ml of *C. burnetii* LPS.

### **CONFOCAL MICROSCOPY**

Cells were fixed with 3% paraformaldehyde in phosphatebuffered saline (PBS pH 7.4) and prepared for immunofluorescence labeling, as previously described (Forestier et al., 1999; Chu and Ng, 2004; Ghigo et al., 2010). Coverslips were mounted in Mowiol, and the cells were imaged using an inverted Leica TCS SPE confocal laser-scanning microscope (Leica, Heidelberg, Germany). Image acquisition was performed using the Leica Confocal software. The collected images were processed using Adobe Photoshop CS5 software. The cells were evaluated as follows: twenty-five fields containing at least three cells per field were examined for each experimental condition; in total, approximately 100 cells were examined per experimental condition, as described elsewhere (Barry et al., 2012). TLR distribution at the cell surface and colocalization analyses were performed using ImageJ software (http://rsb*.*info*.*nih*.*gov/ij) (Bolte and Cordelieres, 2006; Barr et al., 2008). In certain experiments, morphological changes in BMDMs challenged or not challenged with *C. burnetii* LPS (1μg/ml) were evaluated as previously described (Honstettre et al., 2004).

### **P38α MAPK PHOSPHORYLATION ASSAY**

The phosphorylation of p38 was assessed using phospho-p38 MAPK cell-based ELISA (R&D Systems) (Boucherit et al., 2012) following the manufacturer recommendations.

### **IMMUNOPRECIPITATION**

BMDMs were treated with or without LPSs (1μg/ml) for 30 min and then lysed with 1% Triton X-100 in a buffer consisting of 10 mM Tris-HCl pH 7.4, 150 mM NaCl, and 1 mM EDTA pH 8.0. TLR-4 (bdbiosciences) was immunoprecipitated via overnight incubation of the total protein with the anti-TLR-2 antibody (bdbiosciences) followed by incubation with protein ASepharose beads (Roche). The immunoprecipitated pellets were washed and analyzed via immunoblotting on 6% polyacrylamide gels using anti-TLR-2 and anti-TLR-4 antibodies. The detection of TLR-2 from the input sample was performed using 50μg of protein. The immunoblots were visualized using an LAS 4000 camera system (GE Healthcare) or an Amersham Biosciences revelator. In some experiments, macrophages were treated with 1μg/ml of cytochalasin D (Sigma–Aldrich) as previously described (Meconi et al., 1998; Honstettre et al., 2004).

### **STATISTICAL ANALYSIS**

The results are expressed as means ± SD and were analyzed using the non-parametric Mann–Whitney *U-*test. Differences were considered significant at *p <* 0*.*05.

# **ACKNOWLEDGMENTS**

We are grateful to L. Alexopoulou (CIML, Marseille, France) for providing the TLR4 knockout mice. This work was supported by the CNRS (PICS 2012-2014 to Eric Ghigo) and by a PQR Campagnia FSE 2007-2013 (Project CREME). Nicolas Boucherit is a fellow of the French Ministry for Research and Technology. Filippo Conti is a fellow of the Scientific Cooperation Foundation "Infectiopole Sud." Veronica Baldassarre is a fellow of the ERASMUS programme. The funding sources had no role in the study design, data collection and analysis, decision to publish, or manuscript preparation.

# **REFERENCES**

**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.

*Received: 04 November 2014; accepted: 10 December 2014; published online: 06 January 2015.*

*Citation: Conti F, Boucherit N, Baldassarre V, Trouplin V, Toman R, Mottola G, Mege J-L and Ghigo E (2015) Coxiella burnetii lipopolysaccharide blocks p38***α***-MAPK activation through the disruption of TLR-2 and TLR-4 association. Front. Cell. Infect. Microbiol. 4:182. doi: 10.3389/fcimb.2014.00182*

*This article was submitted to the journal Frontiers in Cellular and Infection Microbiology.*

*Copyright © 2015 Conti, Boucherit, Baldassarre, Trouplin, Toman, Mottola, Mege and Ghigo. 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.*

# *Leishmania* lipophosphoglycan: how to establish structure-activity relationships for this highly complex and multifunctional glycoconjugate?

#### *Claire-Lise Forestier 1 \*, Qi Gao2 and Geert-Jan Boons 2*

*1 INSERM U1095, Faculté de Médecine, University of Aix-Marseille, Marseille, France*

*2 Complex Carbohydrate Research Center, Department of Chemistry, University of Georgia, Athens, GA, USA*

### *Edited by:*

*Eric Ghigo, Centre National de la Recherche Scientifique, France*

### *Reviewed by:*

*Gerald Spaeth, Institut Pasteur, France Stephen Peter Kidd, The University of Adelaide, Australia*

### *\*Correspondence:*

*Claire-Lise Forestier, INSERM U1095, Faculté de Médecine, 27 boulevard Jean Moulin, 13005 Marseille, France e-mail<EMAIL_ADDRESS>A key feature of many pathogenic microorganisms is the presence of a dense glycocalyx at their surface, composed of lipid-anchored glycoproteins and non-protein-bound polysaccharides. These surface glycolipids are important virulence factors for bacterial, fungal and protozoan pathogens. The highly complex glycoconjugate lipophosphoglycan (LPG) is one of the dominant surface macromolecules of the promastigote stage of all *Leishmania* parasitic species. LPG plays critical pleiotropic roles in parasite survival and infectivity in both the sandfly vector and the mammalian host. Here, we review the composition of the *Leishmania* glycocalyx, the chemical structure of LPG and what is currently known about its effects in the mammalian host, specifically. We will then discuss the current approaches employed to elucidate LPG functions. Finally, we will provide a viewpoint on future directions that this area of investigation could take to unravel in detail the biological activity of the specific molecular elements composing the structurally complex LPG.

**Keywords:** *Leishmania* **glycoconjugates, lipophosphoglycan, LPG structure, LPG function, chemical synthesis, LPG structure-activity relationships**

# **THE** *LEISHMANIA* **SURFACE COAT**

Like all the parasites of the Trypanosomatid family, *Leishmania* is characterized by the presence of a glycocalyx covering the entire parasite surface (Ferguson, 1999). The surface coats of these different trypanosomatid parasites exhibit a significant diversity in composition. However, all of the surface-bound molecules of this family share a common structural feature, which is that they all contain a highly conserved glycosylphosphatidylinositol (GPI) anchor motif. Notably, this type of GPI-lipid anchor is unusual and structurally very different from those found in mammalian cells (Mcconville and Ferguson, 1993).

Unlike other trypanosomatids in which the glycocalyx is primarily composed of GPI-anchored glycoproteins, the glycocalyx of the *Leishmania* promastigote stage is dominated by GPI-anchored phosphoglycosylated glycans. Lipophosphoglycan (LPG) represents one of the most abundant promastigotespecific surface glycoconjugates, with approximately 5 × 106 copies/cell (Turco and Descoteaux, 1992). The glycosylinositol phospholipids (GIPLs) also termed free GPI, constitute a complex family of abundant low-molecular-weight molecules, with approximately 107 copies/cell. Three different types of GIPL molecules have been described based on the nature of their glycan moiety (Mcconville et al., 1993). In the GIPL of type 1, the glycan part is structurally similar to that of the LPG glycan core, whereas in the GIPL of type 2, the glycan part is related to that of the GPI-anchored glycoprotein. The GIPLs of type 3 exhibit features of type 1 and 2. The membrane-bound proteophosphoglycans (mPPGs) represent a distinct family of GPI-anchored protein-linked glycans that express a phosphoglycan domain structurally similar to LPG. The mPPGs are significantly expressed at the promastigote parasite surface but to a lesser proportion than LPG and GIPLs (Ilg, 2000). Last, one of the major GPI-anchored glycosylated proteins present at the promastigote plasma membrane is GP63, with around 5 × 105 copies/cell. Importantly, the composition of the *Leishmania* surface glycocalyx changes dynamically during the life cycle of the parasite. When infective, promastigote parasites differentiate into obligate intracellular amastigotes in the infected mammalian host cell, the expression of LPG is drastically downregulated. In contrast, GIPLs and PPGs remain highly expressed throughout the parasite life cycle (Turco and Sacks, 1991). Notably, the PPGs continue to be produced in amastigotes, but as free macromolecules rather than membrane-associated ones (Bahr et al., 1993).

The glycoconjugates of the *Leishmania* promastigote membrane are evenly distributed over the entire parasite surface. They form a highly hydrophilic barrier easily detected as an electrondense material using electron microscopy. Its thickness can reach up to 15 nm due to the length of the LPG polysaccharide chain and potentially up to several hundred nanometers due to the lengths of the mPPGs (Ilg, 2000). Because of their abundance, structural uniqueness and specific distributions, the *Leishmania* membrane glycoconjugates are believed to play important functions in the mammalian host. Among these compounds, LPG has attracted considerable attention because its clear implication in multiple activities that favor parasite virulence.

# *LEISHMANIA* **LPG STRUCTURE**

*Leishmania* LPG, is a highly complex macromolecule composed of four distinct domains: a GPI anchor, a glycan core, a linear phosphoglycan chain (PG) and a terminating oligosaccharide cap (**Figure 1**) (Turco and Descoteaux, 1992). The GPI anchor domain consists of an alkyl phosphatidylinositol having a single saturated C24−26 aliphatic chain (Ferguson, 1999). The LPG glycan core is a heptasaccharide comprising two galactopyranosides, a galactofuranoside (Galf), two mannosides and a glucosamine residue attached to inositol. The glycan core is linked to a linear PG that consists of 15–40 phosphodisaccharide (Galβ1,4Manα1-PO4) units. Finally, LPG is terminated by a di-, tri- or tetrasaccharide consisting of galactose and mannose assembled as Manα1,2Manα1 or as Galβ1,4(Manα1,2)Manα1 depending on the *Leishmania* species.

The lipid anchor, glycan core and the linear PG moieties that constitute the LPG are identical in all *Leishmania* species (Descoteaux and Turco, 1999). Despite conservation of these domains, however, LPG exhibits substantial heterogeneity, with important parasite stage- and species-modifications found in the oligosaccharide cap and in the substituents groups branched on the linear PG (Turco et al., 2001; De Assis et al., 2012). Stagespecific variations are observed throughout the parasite life cycle such that LPG undergoes considerable structural modifications in the PG and the terminating cap during parasite metacyclogenesis (Sacks et al., 1990, 1995). In the PG domain, the number of repeating units increases such that the metacyclic promastigote LPG is significantly longer than the procyclic promastigote LPG. In the oligosaccharide cap, change is made with the replacement of the galactoside residue by an arabinopyranoside residue. Species-specific variations occurring in the PG domain of the LPG are one of the main features of this virulent factor. Three types of LPG have been described depending on the nature of the side chain residues and on the site of substitution occupied by these residues in the PG domain. The LPG of *L. donovani* has no side substitution in the PG and therefore remains linear (Sacks et al., 1995). The LPGs of *L. major*, *L. mexicana, L. infantum*, and *L. tropica* are glycosylated at the C3 position of the galactose in the linear PG (Soares et al., 2002) and the LPGs of *L. aethiopica* are frequently mannosylated at position C2 of the mannose. Additionally, the variability in the sugar residues that branch on the PG domain increases significantly

shown in red. The linear conserved phosphoglycan chain is in blue. The terminating oligosaccharide cap is shown in pink. In each domains, the residues that are not conserved among the LPG of the different Leishmania species are represented in a different color (Man, X, Y and Glc). The Glucose phosphate

substituted with different residues. X represents the substitutent group express in the LPG *L. mexicana*, *L. major* and *L. tropica*. Y is a substitutent present in the LPG of *L. aethopica.* Gal, galactose; Man, Mannose; Glc, glucose; Galf, galactofuranose; GlcN, glucosamine; Ino, inositol.

the level of LPG complexity. Finally, intraspecific LPG variability has been observed among similar *Leishmania* species obtained from different field isolates (Coelho-Finamore et al., 2011). Such stage-specific polymorphisms and intra- and interspecies variations have been involved in the survival of *Leishmania* inside the sand fly, more precisely in the selectivity, permissivity, and competence of a given sand fly vector for particular *Leishmania* strains (Dobson et al., 2006, 2010; Volf et al., 2014). However, the role and the biological relevance of LPG polymorphism in the mammalian host has been poorly understood.

Given the structural complexity and heterogeneity of LPG molecules, the identification of the molecular elements responsible for its biological activity have only partially been resolved.

## **ROLE OF LPG IN** *LEISHMANIA* **INTERACTIONS WITH THE IMMUNE SYSTEM**

The LPG-enriched glycocalyx of *Leishmania* constitutes the primary interface of the host-parasite interactions that take place in the dermis of the mammalian host immediately after parasite inoculation by the sandfly vector. Consequently, LPG is the first target for immune detection and at the same time a barrier protecting the parasite from the attack of the host immune system.

LPG has been shown to circumvent the lysis of the parasite by the host complement system. It acts either by sterically preventing the attachment of complement molecules or by directly inactivating the assembly of a functional complement complex at the promastigote surface (Puentes et al., 1989, 1990).

LPG has been shown to favor intracellular parasite survival by interfering with the pro-inflammatory host cell responses via binding of Toll-like receptor (TLR) 2 and 4 on macrophages and NK cells (Becker et al., 2003; De Veer et al., 2003; Kavoosi et al., 2009; Rojas-Bernabe et al., 2014). LPG-TLR interactions induce ERK phosphorylation while suppressing p38 MAP kinase phosphorylation, modulate the production of reactive oxygen species and nitric oxide and inhibit pro-inflammatory cytokine secretion (Chandra and Naik, 2008; De Assis et al., 2012). These studies showed that the integrity of the lipid anchor as well as the length of the PG domain of LPG are involved in the magnitude of LPGmediated host cell activation via TLR2, as procyclic promastigotes are weaker stimulators than metacyclic promastigotes. The level of LPG expression is also been considered as a critical parameter of this specific host cell stimulation pathway (Srivastava et al., 2013). Given that the GPI anchor of *trypanozoma cruzi* has been implicated in TLR2-mediated activation, it is conceivable than the analogous site in *Leishmania* LPG play a similar function (Campos et al., 2001). Finally, by comparing the LPG from *L. braziliensis* and *L. infantum* a recent study reveals that interspecies LPG structural polymorphism has a significant impact on host cell stimulation via TLR (De Assis et al., 2012; Ibraim et al., 2013) Despite these advances, it remains to be determined which LPG motifs are exactly implicated in TLR binding and further immune cell stimulation.

The role of LPG during parasite internalization and multiplication within the host cell has been extensively studied but they have yielded contradictory results. For instance LPG has been shown to delay the maturation of the parasite-containing phagosome by preventing its fusion with lysosomes while some groups have demonstrated that such phenomenon does not occur (Desjardins and Descoteaux, 1997; Forestier et al., 2011). In parallel, LPG has been found to block the assembly of NADPH oxidase and prevent recruitment of proton ATPases at the phagosomal membrane (Lodge and Descoteaux, 2005; Vinet et al., 2009). This function has been attributed to the localization of LPG at the membrane of the *Leishmania*-containing phagosome (Dermine et al., 2005; Winberg et al., 2009) (**Figure 2**). Therefore investigating a potential correlation between the intracellular localization of LPG inside the host cells and a particular biological function would require to be explored. Since the first observation of the presence of the PG disaccharide repeat units of the LPG at the surface of infected host cells (Tolson et al., 1990), a phenomenon that was later confirmed by our group (Forestier, 2013), the fate and pattern of trafficking of LPG during the infection process remain elusive. Given this lack of knowledge, monitoring LPG trafficking in the host cell and in the host organism during the infection process will be key to better understand LPG functions in its mammalian host. Furthermore, investigating whether chemical modifications of LPG occur during the infection process will help unraveling the importance of the distinct LPG structural motifs on its biological functions.

# **LPG AS A PROMISING VACCINE CANDIDATE**

Despite its lack of strong immunogenicity but because of its unique structure, dense distribution and accessibility, *Leishmania* LPG has been considered as an attractive vaccine target (Goel et al., 1999). Early vaccine studies indicated that purified LPG provides protection in mice against challenge with virulent parasites. These studies have demonstrated that LPG-mediated protection can be obtained with LPG alone and that its efficacy depended on the integrity of the LPG molecule and could be modulated by the use of adjuvants (Handman and Mitchell, 1985; Russell and Alexander, 1988; Mcconville and Ferguson, 1993;

**FIGURE 2 | Localization of LPG at the membrane of the** *Leishmania***-containing phagosome.** Bone marrow macrophages were infected with *L.donovani* promastigote for 1 h at 37◦C then macrophages were washed to remove extracellular parasites and incubated in new medium for 24 h. Infected cells were processed for immunofluorescence staining. The LPG molecules were stained using the anti-PG antibody CA7AE (Red), the lysosome and phagosome compartments were stained using the anti-LAMP-1 antibody (Green), the host cell and the parasite nuclei were stained using the Hoechst dye (Blue). Images are analyzed using the Imaris software. **(A)** Represents a single Z section of 0.7μm. **(B)** Represents a zoomed image area of **(A)**. Panel **(C)** is a 3D reconstruction of the entire Z stack. Arrow points to two Leishmania-containing vacuoles showing LPG at the phagosomal membranes. Scale bar, 10μm.

Karanja et al., 2011). Contradictory results have arisen in more recent studies, in both protective and disease-promoting effects associated with LPG vaccination have been observed. These studies have revealed the importance of the immunization route in the vaccine outcome, with subcutaneous LPG injection failing to protect mice against *L. amazonensis* but intranasal administration of LPG showing to be protective (Pinheiro et al., 2005, 2007). Although the mechanisms of LPG-mediated immunization are yet not known, LPG has been shown to activate T cells and favor a Th1 immune response thus mediating protection against the intracellular stage of *Leishmania* (Handman and Mitchell, 1985; Moll et al., 1989; Moll and Rollinghoff, 1991; Tonui et al., 2003; Amprey et al., 2004). Paradoxically, a recent study showed that LPG vaccination, depending on the dose of LPG, induces the expression of the inhibitory receptors PD-1 and PD-L2 on T cells and macrophages respectively, therefore preventing proper protection against leishmaniasis (Martinez Salazar et al., 2014). Given the structural complexity of LPG, it remains unknown which feature of this glycoconjugate, independently or as a part of the whole macromolecule, is involved in the effective immunization process. Therefore, it is important to identify the functionally relevant molecular elements of LPG with the goal of developing artificial and well-designed LPG-based vaccines (Routier et al., 1999; Hewitt and Seeberger, 2001; Astronomo and Burton, 2010; Topuzogullari et al., 2013).

### **APPROACHES TO ASSESS LPG FUNCTIONS**

The involvement of *Leishmania* LPG in virulence has been confirmed in non-physiological conditions, using purified LPG molecules tested on macrophages *in vitro* and, in a more biologically relevant context, using parasites defective in specific steps of the LPG biosynthesis pathway.

Although the use of purified LPG has been very valuable to unravel LPG functions, it also has several drawbacks. Among these is the complicated purification procedures that it requires and along with it the difficulty of obtaining pure LPG preparations devoid of various contaminants. More problematic is the possibility that LPG preparation may become contaminated with trace amount of endotoxin, a problem commonly faced with the purification of molecules and that will greatly bias the host cell immune response. Finally, the use of purified LPG does not reflect the physiological conditions experienced in host cellparasite interaction, and the dose of LPG used in such artificial functional assays may not replicate that encountered in actual physiological conditions.

Genetic approaches rely on the identification of genes encoding for enzymes that are involved in LPG biosynthesis, on the disruption of these target genes in *Leishmania* and on the analysis of the phenotypes and functions of such null mutants (Beverley and Turco, 1998). This type of studies has led to the generation of parasites displaying LPG molecules that are truncated at different levels of their polysaccharide moieties. *Leishmania* parasites were generated with mutations in the LPG1, LPG2, LPG5, and LPG3 genes that, respectively, encodes a galactofuranosyltransferase involved in the synthesis of the LPG glycan core specifically (Ryan et al., 1993), a Golgi GDP-mannose transporter required for the synthesis of the PG domain common to LPG and mPPG (Ma et al., 1997), a Golgi UDP-Gal transporter critical for the synthesis of the PG (Capul et al., 2007a,b) and the *Leishmania* homolog of a mammalian endoplasmic reticulum chaperone required for complete PG synthesis (Descoteaux et al., 2002). As a result, lpg1− parasites express intact GIPLs and mPPG molecules, whereas LPG molecules have a truncated glycan core and no PG domain neither terminal oligosaccharide cap (Spath et al., 2000). In contrast, lpg2− and lpg5− parasites express normal GIPLs but these parasites lack all PG domains including those of LPG and mPPG; however LPG molecules have a normal glycan core (Spath et al., 2003; Liu et al., 2009). Finally, lpg3− parasites express LPG molecules truncated after the first mannoside residue of the first disaccharide unit composing the PG domain (Descoteaux et al., 2002).

These null-mutant parasites and others recent LPG-mutants (Phillips and Turco, 2014) provide powerful tools for identifying the functions of the LPG in a context that closely mimics the natural course of infection, including a physiological concentration of LPG interacting with the host. Importantly, analyses of these mutants have been proven to discriminate efficiently between the roles of LPG and those of other related glycoconjugates, including mPPG, that express similar polysaccharide domains. Nevertheless, the nature of the lpg1, lpg2, lpg3, and lpg5 mutants offers the possibility of assessing the biological activity of only the LPG polysaccharide domains including the last three sugar residues of the glycan core, the phosphoglycan disaccharide repeating units and the oligosaccharide cap. To the best of our knowledge, the functional impact of the species-specific substituents of the PG domain and the nature and structure of the GPI anchor has not been yet investigated. Most likely, this reflects the difficulty inherent in engineering parasites deficient in such specific and essential structural motifs. Indeed, such an approach requires first the identification of the specific genes involved in the addition of the substituent to the linear PG and involved in GPI-anchor biosynthesis and then the mutation of these genes, to obtain viable parasites expressing modified glycoconjugates. Previous attempts to generate GPI-null *Leishmania* have demonstrated that this domain is critical for parasite viability and infectivity (Ilgoutz et al., 1999; Garami et al., 2001). In contrast, parasites deficient exclusively in the assembly of the GPI-anchored glycoproteins but not in the expression of LPG or GIPLs retain their capacity to grow and remain virulent (Mensa-Wilmot et al., 1994; Hilley et al., 2000; Zufferey et al., 2003). However, these studies were not able to discriminate between the GPI-anchor of LPG and other related structures carried by the other glycoconjugates featuring the *Leishmania* surface. To achieve a complete map of LPG structure-function relationships, it is critically required to identify genes involved specifically in the different steps of LPG biosynthesis, so that new LPG mutants may be generated.

Nevertheless, despite the valuable information gained by these studies, the main limitation of such genetic approaches is the limited opportunity they afford to investigate in detail and independently the relative roles of the different structural motifs of highly complex LPG molecules (oligosaccharide cap, phosphopolysaccharide chain, glycan core, GPI anchor and fatty acid chain). Although LPGs display high levels of heterogeneity, whether the molecular composition of the LPG motifs plays a distinct role in LPG function has never been determined. Therefore, there is an urgent need to explore the relative implications and contributions of the different LPG molecular elements to its biological activity.

# **FUTURE APPROACHES**

In our point of view, one of the priorities for future research into the functions of LPG is to elucidate the structure-activity relationship of this membrane-bound glycoconjugate. The ultimate goal is to identify which LPG motifs are associated with specific effects in the mammalian host. Such informations are expected to bring key new insights into the mechanism of action of this macromolecule.

To overcome some aspects of the limitations linked to the genetic approaches, alternative methods that will aim to dissect the LPG functional groups at the molecular level will need to be developed. Chemical synthesis of such structurally complex glycoconjugates may be one of the promising experimental approaches for investigating the distinct functions of each of the structural elements that compose the multifunctional LPG molecules. With technological advances in chemistry, new synthetic strategies and methods for the chemical synthesis of highly complex glycoconjugates are currently developed which will make conceivably the synthesis of LPG feasible (Astronomo and Burton, 2010). These chemical synthesis methods will allow the design and production of panels of synthetic LPG variants having independent molecular variations within its four distinct domains. Such synthetic LPG compounds will be crucial tools for investigating the functional relevance of the molecular elements of LPG. Using chemical synthesis one could expect to address and elucidate the importance of (i) the composition of the fatty acid chain of the GPI-anchor; (ii) the glycan core; and (iii) the repetitive units in the PG domain. Comparisons among the chemically well-defined collection of LPG variants will allow us to assign a molecular motif to a biological function. Such chemical synthesis methodology-based research could be extended to the study of all the others GPI-anchored glyconconjugates expressed at the cell surface of *Leishmania* and others related parasites. This will include GIPLs that unlike LPG, is expressed both at the promastigote and amastigote parasite stages and for which information about its role in the mammalian host remains very limited.

This type of approach will significantly advance our knowledge of the structurally complex LPG. Significantly, it will help to dissect the various LPG domains and attribute a precise function to each specific LPG elements, thereby establishing a causal structure-activity relationship. Ultimately, such chemistry-based strategy will open new venue to the development of LPG-based therapeutics agents against Leishmaniasis using synthetic and biologically relevant molecular elements of LPG.

### **ACKNOWLEDGMENTS**

We acknowledge financial support from the human frontier science program organization (HFSPO), from the INSERM Institute and Support by the National Institute of General Medical Sciences (R01GM061761) of the US National Institutes of Health. I apologize to colleagues whose studies have not been cited here due to space restrictions.

### **REFERENCES**

fly vector requires a specific SCG-encoded lipophosphoglycan galactosylation pattern. *PLoS Pathog.* 6:e1001185. doi: 10.1371/journal.ppat.1001185

response: a model to assess vaccine efficacy. *Vaccine* 32, 1259–1265. doi: 10.1016/j.vaccine.2014.01.016

**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.

*Received: 30 October 2014; accepted: 22 December 2014; published online: 21 January 2015.*

*Citation: Forestier C-L, Gao Q and Boons G-J (2015) Leishmania lipophosphoglycan: how to establish structure-activity relationships for this highly complex and multifunctional glycoconjugate? Front. Cell. Infect. Microbiol. 4:193. doi: 10.3389/fcimb. 2014.00193*

*This article was submitted to the journal Frontiers in Cellular and Infection Microbiology.*

*Copyright © 2015 Forestier, Gao and Boons. 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.*

# Granulomatous response to *Coxiella burnetii*, the agent of Q fever: the lessons from gene expression analysis

#### *Delphine Faugaret 1,2, Amira Ben Amara1, Julie Alingrin1, Aurélie Daumas 1, Amélie Delaby1,3, Catherine Lépolard1, Didier Raoult 1, Julien Textoris 1,4 and Jean-Louis Mège1 \**

*1 Aix Marseille Université, URMITE, UMR CNRS 7278, IRD 198, INSERM 1095, Marseille, France*

*2 AltraBio SAS, Lyon, France*

*3 Centre d'Immunologie de Marseille-Luminy, Parc Scientifique et Technologique, Marseille, France*

*4 Unité Mixte BioMérieux-HCL, Hôpital Edouard Herriot - Pav P, Lyon, France*

### *Edited by:*

*Benjamin Coiffard, Hôpital Nord, France*

### *Reviewed by:*

*Janakiram Seshu, The University of Texas at San Antonio, USA Subramanian Dhandayuthapani, Texas Tech Health Sciences Center, USA*

### *\*Correspondence:*

*Jean-Louis Mège, Aix Marseille Université, URMITE, UMR CNRS 7278, IRD 198, INSERM 1095, 27 Bld. Jean Moulin, 13005 Marseille, France*

*e-mail<EMAIL_ADDRESS>
The formation of granulomas is associated with the resolution of Q fever, a zoonosis due to *Coxiella burnetii*; however the molecular mechanisms of granuloma formation remain poorly understood. We generated human granulomas with peripheral blood mononuclear cells (PBMCs) and beads coated with *C. burnetii*, using BCG extracts as controls. A microarray analysis showed dramatic changes in gene expression in granuloma cells of which more than 50% were commonly modulated genes in response to *C. burnetii* and BCG. They included M1-related genes and genes related to chemotaxis. The inhibition of the chemokines, CCL2 and CCL5, directly interfered with granuloma formation. *C. burnetii* granulomas also expressed a specific transcriptional profile that was essentially enriched in genes associated with type I interferon response. Our results showed that granuloma formation is associated with a core of transcriptional response based on inflammatory genes. The specific granulomatous response to *C. burnetii* is characterized by the activation of type 1 interferon pathway.

**Keywords: granuloma, Q fever,** *Coxiella burnetii***, BCG, transcriptome, type 1 interferon pathway**

# **INTRODUCTION**

Q fever is a worldwide zoonosis caused by *Coxiella burnetii* (Mege et al., 1997). The primary *C. burnetii* infection leads to isolated fever, pneumonia, or hepatitis in 40% of exposed individuals. *C. burnetii* infection may become chronic in patients with valvular lesions, pregnant women, or immuno-compromised patients. In contrast with acute Q fever where the outcome is usually favorable, chronic Q fever is characterized by a long-term drug treatment and persistent risk of relapses. Interestingly, tissue granulomas are present in patients with acute Q fever. In chronic Q fever, granulomas are absent, replaced by lymphocyte infiltrates (Raoult et al., 2005), suggesting that granulomas play an important role in the resolution of Q fever.

Granulomas, defined as tissue collections of macrophages, are generated in response to various microorganisms (Zumla and James, 1996). Their organization varies according to the type of microorganism. In humans, *C. burnetii*-induced granulomas, which are paucibacillary, are composed of a lipid vacuole surrounded by a fibrinoid ring, the "doughnut granuloma" (Srigley et al., 1985; Travis et al., 1986). In contrast, tuberculous granulomas, which are multibacillary, consist of a necrotic core containing bacilli, enclosed by macrophages and surrounded by lymphocytes (Ulrichs and Kaufmann, 2006).

Granulomas are not static organizations but are characterized by continual remodeling and interactions between cell partners (Ramakrishnan, 2012; Shaler et al., 2013). After initial uptake of microorganisms by resident macrophages, the granuloma formation is initiated by recruiting macrophages and bloodderived myeloid cells. The recruitment of activated T-cells by these nascent granulomas completes granuloma formation, and renders them functional (Egen et al., 2008). The main function of granulomas is to contain infectious agents within a limited area, thus restricting the spread of pathogens. Once the infection is contained, the granuloma cells participate in the destruction of infectious agents. Indeed, wild type mice clear mycobacterial infection through granuloma formation whereas mycobacteria disseminate and granulomas are absent in mice that do not express interferon-γ (Cooper et al., 1993). In the majority of patients with tuberculosis, the presence of calcified granulomatous lesions is associated with a controlled infection (Ulrichs and Kaufmann, 2006). However, in others, mycobacteria induce the necrosis of infected macrophages, resulting in a caseum at the center of granulomas. This accumulation of caseum leads to collapsing granulomas and the spread of bacteria (Russell et al., 2009).

Studying granuloma formation in mice requires invasive methods that are not appropriate for human studies. A method was recently developed to generate human granulomas *in vitro* using peripheral blood mononuclear cells (PBMCs) co-cultured with beads coated with BCG (Puissegur et al., 2004; Delaby et al., 2010) or *C. burnetii* extracts (Delaby et al., 2010). This method enables to follow the initial events of granuloma formation and to investigate the molecular mechanisms of granulomas (Egen et al., 2008; Delaby et al., 2012). In this study, we used a high throughput transcriptomic approach to characterize human granulomas induced *in vitro* by *C. burnetii* and to compare them with those induced by BCG. We found that numerous modulated genes were shared by *C. burnetii*- and BCG-induced granulomas, including chemotaxis-associated genes and M1 genes. *C. burnetii* induced a specific repertoire of upmodulated and downmodulated genes that included the activation of interferon-stimulated genes (ISGs), which confers a new role for this pathway in host response to *C. burnetii*.

### **MATERIALS AND METHODS**

### **PATIENTS WITH Q FEVER**

The study was approved by the Ethics Committee of the Aix-Marseille University. Written informed consent was obtained from each subject. Four patients with acute Q fever and 5 patients with Q fever endocarditis were selected. The diagnostic of acute Q fever was based on serological determination of anti-phase II *C. burnetii* antibodies (Abs). The suspicion of Q fever endocarditis was based on standardized questionnaire that included pathological evidence of endocarditis, positive echocardiograms, positive blood cultures, high titers of IgG specific for phase I *C. burnetii* (Raoult, 2012). The average age of patients with acute Q fever was 43 years old (ranging from 30 to 57 years old). The average age of patients with Q fever endocarditis was 54 years old (ranging from 40 to 74 years old). Six healthy individuals (with a mean age of 37 years, ranging from 28 to 56 years old) were used as controls.

### **PREPARATION OF CIRCULATING CELLS**

PBMCs were prepared from leukopacks (Etablissement Français du Sang) or blood collected in ethylene-diamine-tetraacetic acid (EDTA) tubes from donors and patients after centrifugation through a Ficoll density cushion. Monocytes were isolated from PBMCs by CD14 positive selection using magnetic beads coated with anti-CD14 antibodies (Miltenyi Biotec). CD14+ monocytes were differentiated into macrophages by cell culture (Ghigo et al., 2010). To obtain M1 macrophages, macrophages were stimulated with 20 ng/mL recombinant human IFN-γ (Tebu-bio) for 18 h. M2 macrophages were obtained by incubating macrophages with 10 ng/mL IL-10 or 20 ng/mL IL-4 (R&D Systems) for 18 h.

### *IN VITRO* **GENERATION OF GRANULOMAS**

Granulomas were induced by using two procedures. First, sepharose beads were coated with bacterial extracts from phase I *C. burnetii* or BCG as previously described (Delaby et al., 2010). PBMCs recovered from leukopacks (2 × 106 per assay) were cultured with 800 coated beads for 8–12 days in the presence of mAbs against CCL2 and CCL5 or control isotypes (R&D Systems). Individual granulomas were then collected by micromanipulation and incubated in 2 mM EDTA, allowing cells to dissociate (Delaby et al., 2012). Second, the granuloma formation in patients with Q fever and healthy donors was determined by incubating PBMCs with *C. burnetii* (Puissegur et al., 2004). PBMCs (2 × 106 cells per assay) were cultured with 2 × 107 heatkilled bacteria (100◦C, 1 h) in RPMI 1640 supplemented with fetal calf serum, L-glutamine and antibiotics in 6-well culture plates at 37◦C. Cell aggregation was observed every 2 days under light microscopy, and cells were recovered after 8–10 days when the size of aggregates was the highest.

### **RNA EXTRACTION AND MICROARRAY**

Total RNA was extracted from granuloma cells using the RNeasy Mini kit (Qiagen) and DNAse treatment. The granuloma cell gene expression was analyzed using 45,000 probes microarray chips (4 × 44K whole human genome G4112F, Agilent Technologies) and One-color Microarray Based Gene Expression Analysis kit, as previously described (Ben Amara et al., 2010). Three samples per experimental conditions were included in the analysis. Following array scans, image analysis and correction of intra-array signals were performed with Feature Extraction Software A.<IP_ADDRESS> (Agilent Technologies) using default parameters. Minimum Information About a Microarray Experiment-compliant data are provided in the Gene Expression Omnibus (GEO) (Moal et al., 2013) at the National Center for Biotechnology Information (http://www*.*ncbi*.*nlm*.*nih*.*gov/geo/), and can be accessed with the GEO series accession number (GSE37666).

### **MICROARRAY ANALYSIS**

Raw signal data were normalized with a False Discovery Rate below 0.1 and an absolute fold change (FC) value of 3.0. All analyses were performed using R software (version 3) with the bioconductor libraries (Gentleman et al., 2004). Functional annotation was performed using ClueGO plug-in (Bindea et al., 2009) and selecting terms belonging to GO Biological (Moal et al., 2013) in Cytoscape software (Smoot et al., 2011). An enrichment/depletion test, along with the Benjamin-Hochberg correction method, was performed for statistic analysis. GO terms from levels 6 to 8 in GO tree were selected, with a kappa score above 0.44, to create functional annotation network. Each node represents a GO term and contains at least 3 genes. The leading group term of a functional group was defined as the group containing the largest number of genes. Identification of biological groups depicted in the pie chart was established by manual selection of articles from Pubmed database, filtered to show those describing gene functions in immune cells. The identification of M1 and M2 signatures in granuloma cells was performed using the gene profiles of macrophages stimulated with IL-4, IL-10, and IFN-γ referenced in Gene Expression Omnibus database (GSE36537).

### **REAL-TIME QUANTITATIVE RT-PCR (qRT-PCR)**

Reverse transcription of 100 ng of total RNA was performed as previously described (Ben Amara et al., 2010). Primers were designs using Primer3 (Moal et al., 2013) and their sequences were listed in Table 1. Quantitative PCR was performed with Light Cycler Fast Start DNA masterPLUS SYBR Green I (Roche applied Science). The results were normalized with the housekeeping gene β-actin. The FC of target genes relative to β-actin was computed using the formula FC = 2−*--*Ct, where *--*Ct = [(CtTarget− CtActin)stimulated− (CtTarget− CtActin)unstimulated] (Moal et al., 2013). The agreement between qRT-PCR and microarray data was assessed by Pearson correlation coefficient.

### **STATISTICAL ANALYSIS**

Comparisons between two groups were performed using the Mann-Whitney *U*-test.

# **RESULTS**

### **GLOBAL GENE PROGRAM OF GRANULOMA CELLS**

We previously reported that PBMCs are capable to form granulomas when they are incubated with beads coated with bacterial extracts (Delaby et al., 2012). We wondered if granuloma formation was associated with specific changes in gene expression programs. We compared the transcriptional profiles of granuloma cells with that of naïve PBMCs using whole genome microarrays. Correspondence analysis was conducted to assess the reproducibility of data. The first axis of variance showed large differences between PBMCs and granuloma cells, while the second axis of variance revealed smaller differences between granuloma cells generated in response to *C. burnetii* and BCG (**Figure 1A**). Hierarchical clustering also showed that the transcriptional responses of granuloma cells were different from that of PBMCs (**Figure 1B**). Most probes that were upmodulated (1337) and downmodulated (1183) were common in *C. burnetii* and BCG challenges. The probes that were specifically modulated by *C. burnetii* included 524 upmodulated probes and 530 downmodulated probes. Conversely, 345 and 247 probes were upmodulated and downmodulated, respectively, by BCG. The expression of only one probe, the *CD163* gene, increased after *C. burnetii* challenge but decreased after BCG challenge (**Figure 1C**). These data showed that about 60% of modulated genes were shared by granuloma cells but that the granulomatous responses to *C. burnetii* and BCG were, in part, specific.

### **M1/M2 POLARIZATION OF GRANULOMA CELLS**

The macrophages are known to be polarized into M1 or M2 cells, which is associated with microbicidal response or permissive response for intracellular bacteria respectively (Benoit et al., 2008). We wondered whether granuloma macrophages, which represent about 40% of granuloma cells (Delaby et al., 2012), were polarized. Nine M1- and 9 M2-related genes (corresponding to 12 and 17 probes, respectively) were selected according to published data (Martinez et al., 2006). Hierarchical clustering analysis showed that granuloma cells clustered with IFN-γ-stimulated macrophages (M1 macrophages) but not with IL-4- or IL-10-stimulated macrophages (M2 macrophages) (**Figure 2A**), demonstrating that granuloma cells exhibited inflammatory/microbicidal phenotype. This finding was confirmed by the measurement of inflammatory cytokines, TNF and IFN-γ, which were released after 9 days (IFN-γ: 204 ± 53 pg/ml; TNF: 1250 ± 250 pg/ml). However, the granulomatous responses to *C. burnetii* and BCG included some features of M2 macrophages. Indeed, *C. burnetii* upmodulated the expression of *CCL23* and *CCL13* genes, and BCG that of *FN1* and *SLCA47* genes. Other differences were evident: *C. burnetii* caused a slight increase in the expression of *TNF* and *EDN1* genes, but increased greatly the expression of *HESX1* and *CXCL9* genes, in comparison with BCG. To confirm microarray results, qRT-PCR was performed on M1 genes (**Figure 2B**). The profiles of each gene were significantly correlated in qRT-PCR and microarray (*R*2 = 0*.*64, *p <* 0*.*008). In addition, the expression of *HESX1* and *TNFSF10* genes was greater in *C. burnetii*-induced granulomas than in BCG-induced granulomas. Conversely, BCG induced a higher increase in the expression of *IDO1* and *TNF* genes

BCG-induced granulomas were clearly separated from PBMCs. **(B)** The hierarchical clustering analysis showed specific clusters of upmodulated and downmodulated probes in granuloma cells compared to PBMCs. Gene expression level is color-coded from blue (downregulation) to red (upregulation). **(C)** The Venn diagram represents the expression of *(Continued)*

### **FIGURE 1 | Continued**

upmodulated and downmodulated probes in *C. burnetii* and BCG granuloma compared with PBMCs. The numbers in overlapping regions indicate the number of probes commonly modulated in granuloma cells obtained in response to *C. burnetii* and BCG. The numbers in non-overlapping regions indicate the number of probes specifically modulated in response to *C. burnetii* or BCG.

than *C. burnetii*. Taken together, these results suggest that granuloma macrophages were rather polarized into M1 macrophages, with subtle differences between *C. burnetii*- and BCG-induced granulomas.

## **FUNCTIONAL ANNOTATION OF** *C. BURNETII***-SPECIFIC TRANSCRIPTIONAL PROGRAM**

The gene expression program specifically induced by *C. burnetii* was analyzed by retaining only genes differentially modulated between *C. burnetii* and BCG (absolute *FC >* 3.0). We found that 206 genes were specifically modulated, and their roles were studied using GO Biological Process annotation and according to immune function. Nearly 50% of them were related to inflammatory mediators (17%), microbicidal activity (12%), anti-inflammatory mediators (9%) and pathogen recognition (8%). Other cell functions, such as chemotaxis, cell death, and metabolism were also differentially modulated (**Figure 3A**). Taken together, the genes that were specifically modulated by *C. burnetii* were organized into functional networks, suggesting a role in granuloma function.

Next, we investigated how these 206 genes were differently modulated in response to BCG or *C. burnetii* relative to PBMCs. A number of genes involved in chemotaxis were similarly modulated in granuloma cells in response to BCG or *C. burnetii* (**Figure S1**). The genes involved in chemotaxis play a critical role in granuloma formation. This is illustrated by the inhibition of granuloma formation when *C. burnetii*-coated beads were incubated with PBMCs in the presence of mAb directed against CLL2 and CCL5 but not with control isotypes. The mAb directed against CCR5 did not affect granuloma formation (**Figure 3B**). The functional groups associated with inflammation were differently modulated in granulomas induced by *C. burnetii* or BCG. Many genes related to inflammation were downmodulated by *C. burnetii* and upmodulated by BCG. The genes belonging to anti-inflammatory mediators were weakly upmodulated in response to *C. burnetii* but were strongly downmodulated by BCG. Nevertheless, *C. burnetii*-induced granulomas were not only characterized by anti-inflammatory program; indeed, *C. burnetii* did induce several genes related to inflammation such as *TNFSF13*, *CH25H*, and *IRF7* genes. The expression of genes related to cell death was also decreased in *C. burnetii*-generated granulomas, but increased in BCG-induced granulomas. Finally, *C. burnetii* strongly upmodulated the expression of genes involved in microbicidal response and, especially, ISGs including *MX1*, *MX2*, *IFI44*, *IFI6*, *IFIT1*, *IFITM2*, *IFITM3*, *ISG15*, *OAS1*, *OAS2*, *OAS3*, and *HERC5* genes, whereas BCG had little effect on these genes. The transcriptional differences between granulomas induced by *C. burnetii* and BCG compared with PBMCs were confirmed by qRT-PCR performed on several genes. Indeed, the

**FIGURE 2 | Atypical M1 polarization of granuloma cells.** The M1/M2 polarization of granuloma cells was studied using macrophages stimulated with IFN-γ (M1), IL-10, IL-4 (M2) as controls of M1 and M2 polarization. Microarrays **(A)** and qRT-PCR **(B)** were performed on M1/M2 macrophages and granuloma cells. **(A)** The hierarchical clustering of specific markers of M1 or M2 polarization indicates that granuloma cells induced by *C. burnetii* and BCG were located within a unique cluster near M1 macrophages. Note that M2 genes were modulated in granuloma cells as well. Gene expression level is color-coded from blue (downregulation) to red (upregulation). **(B)** The expression of *HESX1, TNFSF10, IDO1,* and *TNF* (M1 genes) was increased in *C. burnetii*- and BCG-induced granulomas, but their expression was differentially modulated in both types of granulomas. ∗*p <* 0*.*05 for the comparison between *C. burnetii* and BCG using Mann-Whitney *U*-test. Mϕ, Macrophages; NS, unstimulated.

expression of genes encoding inflammatory mediators such as *EPHB2* gene and *EDN1* gene was highly increased in response to *C. burnetii* and BCG, respectively. The expression of genes involved in cell death, such as *FASLG* and *GNLY*, was increased

in response to BCG. In contrast, the expression of ISGs (*IFIT1* and *OAS2*) was highly upmodulated in granulomas induced by *C. burnetii* (**Figure 3C**). Taken together, these results showed that the granulomatous response shared common features, but also included specific characteristics, according to the nature of the pathogen. They also showed that the granulomas induced by *C. burnetii* were characterized by the activation of type 1 IFN genes.

### **TRANSCRIPTIONAL RESPONSE OF GRANULOMAS IN Q FEVER PATIENTS**

We finally inquired whether the granulomatous response of patients with Q fever was associated with alteration in type 1 IFN pathway. First, we developed a simple method to obtain granulomas by incubating control PBMCs with *C. burnetii* to simplify the recovery of isolated granulomas using *C. burnetii*-coated beads. Cell aggregates were observed after a few days. Their size progressively increased and was greatest at 8–10 days, demonstrating that the time course of granuloma formation was similar when granulomas were generated *in vitro* using beads coated with bacterial extracts (Delaby et al., 2012) or heat-killed bacteria. We investigated the expression of *IFIT1* and *OAS2*, two genes belonging to type 1 IFN pathway which were specifically modulated in *C. burnetii*-induced granulomas. The expression of *IFIT1* (**Figure 4A**) and *OAS2* (**Figure 4B**) genes was strongly upmodulated in stimulated PBMCs, but not in unstimulated PBMCs. We noted that the levels of expression of *IFIT1* and *OAS2* genes were similar to those obtained with isolated granuloma cells (see **Figure 3C**). The expression of *IFIT1* genes (**Figure 4C**) and *OAS2* genes (**Figure 4D**) was similar in *C. burnetii*-induced granulomas from patients with Q fever and healthy controls. These results demonstrated that type 1 IFN pathway was not altered in Q fever granulomas.

# **DISCUSSION**

The favorable outcome of Q fever is associated with the presence of granulomas (Raoult et al., 2005), but the real functions of granulomas during *C. burnetii* infection remain unknown. Therefore, we employed a technique that generated *in vitro* human granulomas (Delaby et al., 2010), then performed whole genome transcriptional profile of *C. burnetii*-induced granulomas and we compared it to that induced by BCG.

More than 50% of genes that were modulated in granulomas, were commonly modulated by *C. burnetii* and BCG. First, they included genes involved in chemotaxis, especially those related to the recruitment of monocytes and lymphocytes, such as *CCL2* (Loetscher et al., 1994), *CCL8* (Loetscher et al., 1994), *CCL13* (Garcia-Zepeda et al., 1996), *CCL17* (Cronshaw et al., 2006), and *CCL18* (Adema et al., 1997) genes. This finding is consistent with the critical role of the recruitment of monocytes and lymphocytes

in *in vitro*-generated granulomas (Delaby et al., 2012). Our data extend to human granulomas the role of chemokines initially described in animal models (Chensue, 2013). Hence, in mice deficient for CCR2, a receptor for CCL2 and CCL8, the number and size of granulomas, as well as monocyte recruitment at site of infection, are decreased (Jinnouchi et al., 2003). We provided evidence that the neutralization of CCL2 was sufficient to prevent the formation of granulomas in response to *C. burnetii* and BCG. As reported above in CCR2 deficient mice, it is likely that CCL2 is involved in the initial stages of granuloma formation. Similar results were obtained with the neutralization of CCL5. This result may be related to the model of CCL5 ko mice in which granuloma function is transiently impaired (Vesosky et al., 2010). In addition, granuloma cells in intestinal tissues from patients with Crohn's disease express CCL5 (Oki et al., 2005). It has been suggested that CCL5 via its interaction with CCR5 augments type 2 granuloma formation (Chensue, 2013). The role of CCL2 and CCL5 in granuloma formation in response to *C. burnetii* is likely not redundant. We hypothesize that a temporal regulation of chemokines is necessary for granuloma formation. Second, granulomas are essentially composed of macrophages, which were similarly polarized in response to *C. burnetii* and BCG. Indeed, granuloma cells expressed M1 profile with some features of M2 cells. This finding is markedly distinct from isolated macrophages infected with *C. burnetii* that express an atypical M2 profile (Benoit et al., 2008). This difference between isolated macrophages and macrophages involved in a functional unit such as granulomas has been reported in mice infected with *Mycobacterium tuberculosis*. Indeed, granuloma-associated macrophages are polarized into M1 cells whereas macrophages surrounding granulomas shift from an M1 to an M2 profile (Redente et al., 2010). In addition, the loss of M2 macrophages during *Leishmania major* infection delays disease progression and increases resistance to pathogens (Hölscher et al., 2006). We hypothesize that the microenvironment of granulomas sustains M1 polarization to eradicate pathogens.

The transcriptional response of *C. burnetii*-generated granulomas was, in part, specific. First, inflammatory genes were differentially modulated. Indeed, *C. burnetii* downmodulated the expression of *TNF*, *NCR3*, *EDN1* and genes related to the Th1 profile, such as *IFNG, TBX21*, which encodes the transcription factor tbet, *IL18RAP*, *IL26*, and *HOPX* genes. Murine macrophages infected with *Leishmania major*, a granulomainducing pathogen, are unable to produce EDN1 (Wahl et al., 2005). A dramatic decrease in inflammatory cytokines (IFN-γ, IL12-p40) was observed in granulomas from BCG-vaccinated guinea pigs induced by *M. tuberculosis* (Ly et al., 2007). Second, *C. burnetii* specifically induced the expression of *CH25H*, *TNFSF13*, and *IRF7* genes, known to be related to type 1 IFN pathway (Park and Scott, 2010; Tezuka et al., 2011) and likely involved in microbicidal response of granuloma cells. For instance, CH25H is an enzyme involved in production of oxysterol, 25-hydroxycholesterol. Oxysterols are known to bind liver X receptor (LXR) and its deficiency is associated with loss of microbicidal competence and apoptosis (Joseph et al., 2004). TNFSF13 likely plays a role in Th1 polarization and TNFSF13 deficient mice exhibit defective bacterial clearance (Xiao et al., 2008). Taken together, these results suggest that the activation of inflammatory genes may limit *C. burnetii* infection in specific granulomas directly or indirectly via type 1 IFN pathway.

The granuloma cells induced by *C. burnetii* exhibited upmodulated expression of numerous genes belonging to type 1 IFN pathway; most of these genes were ISGs. Although ISGs have been associated initially with antiviral response (Schoggins et al., 2011), many recent studies report their upregulation in response to bacteria and bacterial components, such as lipopolysaccharide (Textoris et al., 2012). Type 1 IFN signaling has also been implicated in efficient clearance of group B *Streptococcus*, *S. pneumoniae*, and *Escherichia coli* (Textoris et al., 2012). Indeed, injection of *C. burnetii* components in mice induces a transient IFN-α/β production, and a steady synthesis of OAS for several days (Zvilich et al., 1995). This finding is potentially important for the defense against *C. burnetii* since type 1 IFN was either not detected or its pathway was defective in macrophages or dendritic cells stimulated by *C. burnetii*, respectively (Gorvel et al., 2014). This may be related to previous reports, which state that a BCG mucoprotein is unable to induce the expression of interferoninducible antiviral proteins (Ishii et al., 2005). The stimulation of type 1 IFN pathway (*OAS2* and *IFIT1* genes) was similar in granulomas from healthy controls or patients with Q fever. ISGs may therefore play an important role in the anti-infectious activity of *C. burnetii* granulomas.

In conclusion, the transcriptional response of *C. burnetii* granulomas includes a common part shared with other infectious granulomas and a part specific for *C. burnetii*. The specific response to *C. burnetii* involved the activation of genes involved in inflammation including type 1 IFN related genes, microbicidal activity, and apoptosis. This transcriptional program may account for the granuloma-mediated elimination of *C. burnetii*, in accordance with the natural history of Q fever where granulomas are associated with a protective immune response and resolution of the disease, whereas the absence of granulomas is associated with the chronic evolution of the disease.

### **ACKNOWLEDGMENTS**

The authors would like to thank Olivia DeLozier for editing the English language manuscript.

### **SUPPLEMENTARY MATERIAL**

The Supplementary Material for this article can be found online at: http://www*.*frontiersin*.*org/journal/10*.*3389/fcimb*.* 2014*.*00172/abstract

### **Figure S1 | Functional annotation of** *C. burnetii***- and BCG-modulated**

**genes.** The expression of the modulated genes in *C. burnetii*- and BCG-induced granulomas was compared to their expression in PBMCs. The functional groups that contain the largest number of distinctly modulated genes were selected, and genes were depicted. The direction of gene regulation is color-coded, with blue indicating downregulation, and red indicating upregulation.

# **REFERENCES**

and disintegration of mycobacterial granulomas. *Immunity* 28, 271–284. doi: 10.1016/j.immuni.2007.12.010

**Conflict of Interest Statement:** The Guest Associate Editor, Benjamin Coiffard, declares that, despite having collaborated with author, Aurélie Daumas, the review process was handled objectively. 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.

*Received: 29 July 2014; accepted: 21 November 2014; published online: 15 December 2014.*

*Citation: Faugaret D, Ben Amara A, Alingrin J, Daumas A, Delaby A, Lépolard C, Raoult D, Textoris J and Mège J-L (2014) Granulomatous response to Coxiella burnetii, the agent of Q fever: the lessons from gene expression analysis. Front. Cell. Infect. Microbiol. 4:172. doi: 10.3389/fcimb.2014.00172*

*This article was submitted to the journal Frontiers in Cellular and Infection Microbiology.*