Pulmonary viremia was previously demonstrated to be increased in PLWH that had active TB compared to those without TB (Nakata et al., 1997). Our results are consistent with these observations and demonstrate the first preliminary evidence, to our knowledge, that these polymicrobial outcomes in the lung could be modeled in the HIS mouse. The observations that HIV+ cells localized to the sites of interstitial inflammation and Mtb proliferation further suggest potential mechanisms of microbial synergy in the lung. The inflammatory immune response that restrains Mtb propagation in lesions may recruit new targets and establish activation conditions favorable for HIV replication. Likewise, the suppression of immunity due to local effects of HIV infection could promote Mtb proliferation. Importantly, our results suggest that these tissue events of coinfection may differ from those in the periphery, as described in one report of human subjects (Nakata et al., 1997). We observed increased viral replication in the lung and liver in the setting of Mtb co-infection while plasma viral loads were similar among HIV and HIV/TB groups. These differences in viremia between the blood and tissues could suggest that blood-based diagnostics may not fully reflect the in vivo disease process in those with co-infection with regard to the impact on viral load or viral reservoirs.

Multiplex analysis of lung analytes from HIS mice with relapse demonstrated a moderate activation of many cytokines and chemokines including pro-inflammatory molecules. HIV infection is known to promote a pro-inflammatory bias in the infected host (Nou et al., 2016) while several inflammatory mechanisms are associated with increased Mtb pathogenesis (Tobin, 2015; Wallis and Hafner, 2015). In the current study, HIV mono-infection generally presented with only moderate and non-significant activation of cytokines and chemokines in the tissues and plasma compared to non-infected animals. Infection with Mtb in the post-drug relapse phase, however, was associated with pulmonary activation of several pro-inflammatory cytokines and chemokines that have established roles in enhancing HIV replication including IL-1β, IL-6, TNF-α, and CCL-2 (Swingler et al., 1994; Kumar et al., 2013; Nou et al., 2016; Pasquereau et al., 2017). CCL2 was recently shown to further support HIV pathogenesis by promoting virion release from infected host cells (Ajasin et al., 2019). Increased proliferation of HIV in lung of persons with Mtb co-infection has been previously associated with increased TNF-α and CCL2 levels in bronchoalveolar lavage (Mayanja-Kizza et al., 2009; Kumar et al., 2013). Further exploration in HIS models may demonstrate if the production of these pro-viral molecules at sites of Mtb-driven inflammation favor HIV replication through a variety of potential mechanisms including recruitment of inflammatory target cells, direct activation of the HIV LTR, and augmented virion release from productively infected host cells.

Effector cytokines with anti-mycobacterial function (e.g., IFNγ, TNF-α, and IL-17) (Ernst, 2012) were also observed to be moderately activated in the lung microenvironment at relapse. Co-infection with HIV did not significantly reduce the activation of IFN-γ or TNF-α at Mtb regrowth endpoints, suggesting lack of a generalized immune suppression. In contrast, HIV infection reduced production of pulmonary IL-17 following activation by Mtb regrowth. These preliminary results may suggest an interesting candidate mechanism of microbial synergy for further exploration. The Th17 subset of CD4+T cells are very permissive to HIV infection due to lack of RNases (Christensen-Quick et al., 2016). Impairment and depletion of Th17 cells in PLWH is also known to occur in the early stages of infection (El Hed et al., 2010; Prendergast et al., 2010; Murray et al., 2018). IL-17 produced by multiple cell populations is important for host immunity to Mtb (Lockhart et al., 2006; Umemura et al., 2007; Okamoto Yoshida et al., 2010; Torrado and Cooper, 2010; Shen and Chen, 2018) and is elevated in persons with latent TB compared to healthy controls (Coulter et al., 2017; Devalraju et al., 2018). In support of our observations, reduced IL-17 was also associated with TB regrowth in a BALB/c mouse model of post-drug TB relapse (de Steenwinkel et al., 2013).

The overall pattern of cytokine and chemokine activation in the liver was similar, but muted, compared to the lung. This outcome is consistent with the lower pathogen burden in this site of dissemination. Similarly, even fewer significant differences in cytokine and chemokine levels were observed in the plasma at relapse. An interesting exception was the activation of CXCL10 observed in the lung, liver, and plasma of animals in the TB relapse group. CXCL10 has been previously described as a biomarker capable of distinguishing active and latent TB and predicting the risk for TB chemotherapy failure (Hong et al., 2014; Wergeland et al., 2015). In contrast to the observations in human subjects that CXCL10 predicted TB disease state irrespective of HIV status (Wergeland et al., 2015), we observed a significant reduction of CXCL10 in the liver and plasma, but not the lung, of HIS mice in the setting of co-infection. Further studies are needed to validate the biomarker potential of CXCL10 and other immune molecules to inform TB relapse outcomes and identify the effect of HIV on these biomarkers. Our preliminary findings support the potential for this small animal model to reproduce important immune outcomes in a system where mechanistic investigations of the lung and other tissue compartments can be undertaken and therapeutic interventions can be tested.

There are several challenges to use of HIS mice that should be noted despite the obvious advantages for study of HIV and co-infections such as HIV and Mtb. Animals are expensive to generate and maintain, and often require 2 to 3 months for development of immune reconstitution following irradiation and engraftment of human stem cells. The chimeric nature of the system means that immune outcomes include both human adaptive and innate, as well as murine innate, responses. There are additionally suboptimal development of some immune responses such as humoral immunity in HIS mice (Karpel et al., 2015). In the NSG mouse strain used to generate BLT mice in the current study, development of GVHD is a known issue (Greenblatt et al., 2012) that led to losses in animal numbers in the prolonged study design required to reproduce TB treatment and relapse as reported here. Recent regulatory and legal issues have further restricted production of, and funding for, the BLT HIS model in many states and institutions. Nonetheless, the outcomes of the current study are important to guide development of models of co-infection including HIV-associated TB relapse in newer HIS models. Cord blood stem cells can generate HIS mice with similar reconstitution efficiency to the BLT (Wunderlich et al., 2018) while further refinements in these models can expand different immune compartments and reduce development of GVHD (Iwabuchi et al., 2018).

In conclusion, our findings describe a candidate small animal model of TB relapse in the setting of HIV infection that reproduces aspects of clinical disease, including microbial synergy and immune activation, observed in co-infected persons. Long term, HIS mouse models of TB relapse may serve an important pre-clinical role for discovery and translation of therapeutics prior to final evaluation in NHP models. Investigations in models capable of supporting co-infections are critical to improving TB treatment outcomes in HIV+ persons.

#### DATA AVAILABILITY STATEMENT

All datasets generated for this study are included in the article/**Supplementary Material**.

#### ETHICS STATEMENT

The animal study was reviewed and approved by the University of Texas Medical Branch Institutional Animal Care and Use Committee.

#### AUTHOR CONTRIBUTIONS

Investigations into the feasibility and application of the HIS mouse to study TB relapse in the setting of HIV were carried out by MH, TS, RN, KN, SC, JR, ME, JL, BG, and JE. Data generated from these investigations was analyzed by MH, RN, KN, SC, RH, JA, BG, and JE. MH and JE prepared the manuscript and upon careful review by TS, RN, KN, SC, JR, and BG. ME edited the document for submission.

#### FUNDING

This work was supported by grants to BG and JE from the NIH (R56 HL129881 and R01 A1HL129881) and the UTMB Institute for Human Infections and Immunity. Fellowship support for MH was provided through the American Society for Microbiology Robert D. Watkins Graduate Research Fellowship Program. RN and KN were supported by Pre-doctoral Fellowships through the UTMB James W. McLaughlin Endowment.

#### ACKNOWLEDGMENTS

We wish to express our sincere gratitude to Mark Griffin, manager of the UTMB Flow Cytometry and Cell Sorting Core

#### REFERENCES

Facility, for his assistance with flow cytometry experiments. We truly appreciate the team of dedicated individuals affiliated with the UTMB Animal Resource Center for animal husbandry during the lengthy infection studies.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcimb. 2020.00150/full#supplementary-material

liver and thymus humanized mouse model. PLoS ONE 7:e44664. doi: 10.1371/journal.pone.0044664

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

Copyright © 2020 Huante, Saito, Nusbaum, Naqvi, Chauhan, Hunter, Actor, Rudra, Endsley, Lisinicchia, Gelman and Endsley. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) 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.

**111**

# 3D Imaging of the Transparent Mycobacterium tuberculosis-Infected Lung Verifies the Localization of Innate Immune Cells With Granuloma

Gyeong-Yi Kang1,2†, Hyeong-Jun Rhyu3†, Hong-Hee Choi 4 , Sung Jae Shin2,4,5 \* and Young-Min Hyun1,2 \*

*1 Department of Anatomy, Yonsei University College of Medicine, Seoul, South Korea, 2 BK21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, South Korea, 3 Department of Medicine, Yonsei University College of Medicine, Seoul, South Korea, 4 Department of Microbiology, Yonsei University College of Medicine, Seoul, South Korea, 5 Institute for Immunology and Immunological Disease, Yonsei University College of Medicine, Seoul, South Korea*

#### Edited by:

*David Neil McMurray, Texas A& M Health Science Center, United States*

#### Reviewed by:

*Selvakumar Subbian, Public Health Research Institute (PHRI), United States Michiel Van Der Vaart, Leiden University, Netherlands*

#### \*Correspondence:

*Sung Jae Shin<EMAIL_ADDRESS>Young-Min Hyun<EMAIL_ADDRESS>
*†These authors have contributed equally to this work*

#### Specialty section:

*This article was submitted to Microbes and Innate Immunity, a section of the journal Frontiers in Cellular and Infection Microbiology*

> Received: *18 February 2020* Accepted: *22 April 2020* Published: *15 May 2020*

#### Citation:

*Kang G-Y, Rhyu H-J, Choi H-H, Shin SJ and Hyun Y-M (2020) 3D Imaging of the Transparent Mycobacterium tuberculosis-Infected Lung Verifies the Localization of Innate Immune Cells With Granuloma. Front. Cell. Infect. Microbiol. 10:226. doi: 10.3389/fcimb.2020.00226* Using a novel tissue-clearing method, we aimed to visualize the three-dimensional (3D) distribution of immune cells within *Mycobacterium tuberculosis* (Mtb)-infected mice lungs. Ethyl cinnamate-based tissue clearing of Mtb-infected mice lungs was performed to obtain transparent lung samples, which were then imaged using a light sheet fluorescence microscope. Using the 3D images, we performed quantitative analysis of the immune cell population within multiple granulomas. In addition, to compare the data from the tissue clearing method, we performed histopathological and immunofluorescence analyses, and flow cytometry. We then created 3D images of the Mtb-infected lung that successfully demonstrated the distribution of blood vessels, immune cells, and granulomas. Since the immune cells within a granuloma could be separately selected and counted, the immune cell population within a specific lesion could be quantified. In addition, macroscopic analysis, e.g., the size or shape of a granuloma, as well as microscopic analysis could be performed as intact lung samples were used. The use of the tissue clearing method in infected lungs could be a novel modality for understanding the role of the immune system in the pathogenesis of tuberculosis.

#### Keywords: granuloma, innate immunity, lung, tissue clearing, tuberculosis

# INTRODUCTION

Tuberculosis (TB), a pulmonary disease caused by a group of infectious agents named Mycobacterium tuberculosis (Mtb) complex, remains one of the major health concerns worldwide. World Health Organization (WHO) reported that nearly 10.4 million people were infected, and 1.7 million deaths were caused by the contagious disease in 2016 (Floyd et al., 2018). The standard treatment for TB includes multiple anti-TB drugs, i.e., isoniazid, rifampin, ethambutol, and pyrazinamide (Tiberi et al., 2018). However, with the advent of drug-resistant TB, which exhibits resistance against one or more of the drugs in the initial regimen, chemotherapy alone has not been demonstrated to control the disease completely (Tiberi et al., 2018). Therefore, novel therapeutic approaches for TB are required.

In light of this need for a new treatment, several researches have attempted to elucidate the role of the innate immune system in the pathogenesis of the disease. The primary cells involved in the initial immune response against TB infection are macrophages, including resident alveolar and monocyte derived macrophages, and neutrophils (Schlesinger, 1996). The defense mechanism of macrophages against the infective agent is relatively well-known. As Mtb enter the alveoli, alveolar macrophages phagocytose the bacteria and subsequently produce various cytokines, recruiting monocytes from the blood and leading to mobilization of monocyte derived macrophages. This is followed by production of reactive oxygen species (ROS) and apoptosis of the recruited macrophages (Pieters, 2008). Recently, several counteractive actions of Mycobacteria against macrophages have also been found (Lerner et al., 2015). In contrast, several findings have identified a controversial role of neutrophils in regard to their interaction with the pathogen (Lowe et al., 2012). Being the most prominent immune cells in human TB, neutrophils are also the most common leukocytes in human blood (Eum et al., 2010). Animal studies have demonstrated that neutrophils play a crucial role in the initial defense against TB (Pedrosa et al., 2000; Fulton et al., 2002; Sugawara et al., 2004). Recruitment of neutrophils results in decreased colony forming units (CFU) while their depletion causes the opposite (Sugawara et al., 2004). One of the known antibacterial actions of neutrophils is its formation of a DNA scaffold, named neutrophil extracellular traps (NET) that entraps pathogens and facilitates bactericidal actions (Papayannopoulos, 2018). However, it has also been reported that there was no difference in the disease course in granulocyte-depleted mice and normal mice (Seiler et al., 2000). Additionally, the contribution of neutrophils is limited to the early stages of the disease (Petrofsky and Bermudez, 1999; Pedrosa et al., 2000; Sugawara et al., 2004; Zhang et al., 2009). In chronic TB infection, neutrophil depletion was shown to be correlated with a decrease in CFU (Zhang et al., 2009). Data from patients also suggest that higher neutrophil count is associated with worse prognosis in well-established TB (Barnes et al., 1988). To summarize, these findings indicate that the role of neutrophils in the immune response of TB remains to be clarified and studying their functions at different stages of the disease may provide a deeper understanding of the pathogenesis.

One possible approach to interpret the complicated immune reaction is to observe the spatial distribution of the immune cells. It has been reported that Mtb-infected macrophages become highly mobile and egress from the primary granuloma, consequently becoming a source of new granulomas in zebrafish and mouse (Davis and Ramakrishnan, 2009; Cohen et al., 2018). This implies that the physical distribution of Mtbinfected macrophages, or how they translocate out of the granuloma, can affect the progress of the disease. In contrast, the local influx of neutrophils has been correlated with worse prognosis while suppressing it by chemokine modulation has a better outcome (Condos et al., 1998; Nandi and Behar, 2011). These findings show that the course of the disease is closely linked with the distribution of the immune cells, such as neutrophils and macrophages. Traditionally, several modalities, e.g., hematoxylin and eosin (H&E) staining, immunohistochemistry, and fluorescence microscopy, have been used to localize cells in a pathologic condition. However, all these methods provide limited information, since the data are mostly 2D images with the possibility of omitting cells. Recently, total organ imaging enabled by a method that makes an organ transparent, namely tissue clearing, is emerging as a novel technique in various fields of research (Chung et al., 2013; Klingberg et al., 2017). One of tissue clearing methods, PACT (passive clarity technique), has also been used for fluorescencelabeled Mtb in the Mtb-infected mouse lungs (Cronan et al., 2015). In this study, we used the another tissue clearing technique using Ethyl cinnamate (ECi) to visualize immune cells within the pathologic tissue. We used 3D imaging technique of the tissuecleared lung to localize neutrophils and macrophages in Mtbinfected mice lungs. Providing data on the spatial distribution of these immune cells and granulomas, we propose a new modality in understanding the role of the innate immune system in the pathogenesis of TB.

#### METHODS

#### Mice

C57BL/6 mice were purchased from Japan SLC Inc. (Shizuoka, Japan). C57BL/6, CX3CR1-GFP (green fluorescent protein) (Jung et al., 2000) and LysM-GFP (Faust et al., 2000) mice were maintained under specific-pathogen-free conditions at the Avison Biomedical Research Center, Yonsei University College of Medicine. In order to minimize the unintended loss in leukocyte motility or its bactericidal function, heterogenetic (+/–) LysM-GFP and CX3CR1-GFP mice were used in the experiments. All animal experiments were performed with the approval by the animal ethics committee of Yonsei University College of Medicine (2016-0178 and 2018-0218).

#### Infection of Mice

Mice were infected under strict barrier conditions in a BSL-3 facility at the Avison Biomedical Research Center, Yonsei College of Medicine. Briefly, mice were challenged with the pre-calibrated Mtb H37Rv (ATCC 27294) via aerosol using an airborne infection apparatus (Glas-Col, USA), and ∼200 viable bacteria were delivered. Mice at 4 weeks post-infection were used. For the bacterial growth analysis, the lungs were homogenized, and serially diluted samples were plated onto Middlebrook 7H11 agar plates (Becton Dickinson, USA) supplemented with 10% OADC (oleic acid albumin dextrose catalase; Difco Laboratories, USA), 2µg/mL 2-thiophenecarboxylic acid hydrazide (Sigma-Aldrich, USA), and amphotericin B (Sigma-Aldrich). The bacterial colonies were counted after 3–4 weeks of incubation period at 37◦C. The animal ethics committee of Yonsei University

**Abbreviations:** CFU, colony-forming units; ECi, ethyl cinnamate; FACS, fluorescence-activated cell sorting; FBS, fetal bovine serum; FDA, U.S. Food and Drug Administration; GFP, green fluorescent protein; H&E, hematoxylin and eosin; LFMS, light sheet fluorescence microscopy; Mtb, Mycobacterium tuberculosis; OADC, oleic acid albumin dextrose catalase; PBS, phosphatebuffered saline; TB, tuberculosis; WHO, World Health Organization.

College of Medicine approved all of the experimental protocols used (2016-0178 and 2018-0218).

# Histopathological and Immunofluorescence Studies

The naïve and Mtb-infected mice lungs were isolated and fixed in 10% formalin overnight. Fixed lungs were serially dehydrated using ethanol and then infiltrated with paraffin. The lungs were then embedded into the paraffin wax block. Paraffin blocks were sectioned by 4–5µm thickness, and then stained using H&E. Samples were observed using Olympus BX51 microscope (Olympus, Japan). For immunofluorescence imaging, antigen retrieval was performed using Tris-EDTA. Immune cells were labeled using anti-CD3 (T cells; BioLegend, USA), anti-Ly-6G (neutrophils; Biolegend, USA), and anti-F4/80 (macrophages; BioLegend, USA) antibodies and nuclei were stained using DAPI (4',6-diamidino-2-phenylindole). The slides were observed using the LSM700 confocal microscope (Carl Zeiss, Germany). Volocity Software (Quorum Technologies, Canada) software was used for data analysis.

#### Flow-Cytometry

The lung samples were minced into 2-4 mm pieces using scissors. The samples were then incubated in 3 mL of cellular dissociation buffer [RPMI medium; Biowest, France)], which contained 0.1% collagenase type IV (Worthington Biochemical Corporation, USA) and 1 mM CaCl2 and 1 mM MgCl2 for 30 min at 37◦C. The dissociated cells were then filtered using a 40µm cell strainer (BD Biosciences, USA) in RPMI medium supplemented with 2% fetal bovine serum (FBS, Biowest, France), and the erythrocytes were removed by incubating the samples in red blood cell lysis buffer (Sigma-Aldrich, USA) for 3 min at room temperature. Finally, the single cells were washed twice in RPMI medium supplemented with 2% FBS before analysis. For flow-cytometric analysis, cells were first washed with 2% FBS containing PBS and anti-CD16/32 antibodies were treated for blocking function at 4◦C for 20 min. Surface molecules were labeled with fluorochrome-conjugated anti-CD45 (immune cells), anti-Siglec-F (alveolar macrophages), anti-Ly-6G (neutrophils) (BD bioscience, USA), anti-F4/80 (macrophages), anti-CD11b (monocytes/macrophages, granulocytes) (eBioscience, USA), anti-CD64 (monocyte-derived macrophages), and anti-CD11c (monocytes/macrophages) (Biolegend, USA) antibodies and using the LIVE/DEAD Fixable Dead Cell Stain Kit (Molecular Probes, USA) at 4◦C for 30 min. The cells were then washed with PBS, fixed with Intracellular (IC) fixation buffer (eBioscience, USA) for flow cytometry analysis. The cells were subsequently analyzed using a CytoFLEX S Flow Cytometer (Beckman Coulter, USA).

# ECi (Ethyl Cinnamate)-Based Tissue Clearing

LysM-GFP and CX3CR1-GFP mice were used for visualization of the immune cells, and FSD-647 Lycopersicon esculentum (L. esculentum) lectin (BioActs, Korea) was intravenously injected to label glycocalyx in the basal membrane of the endothelial cells. ECi clearing method was selected as it has benefits such as minimizing sample shrinkage and preserving endogenous fluorescence for fluorescence imaging (Klingberg et al., 2017). In addition, the method is not toxic, and has been approved by the U.S. Food and Drug Administration (FDA). The aqueous buffer clearing method was excluded as it can make samples inadequately clear and may cause the samples to swell giving them a jelly-like texture. Before conducting tissue clearing using the ECi solution, the mice were perfused with 1X PBS and 4% formaldehyde via the myocardial route. Perfusion using this method enables effective fixation of mice lungs by utilizing the circulatory system of the animal. The intact lungs were then fixed in 4% formaldehyde solution for 2 h and dehydrated in alkaline solutions for 12 h (50/70/100% ethanol, 4 h each). Finally, the samples were cleared using the ECi solution (Alfa Aesar, USA) for 30 min at room temperature. All incubation processes were conducted away from light. The cleared samples were stored in polypropylene tubes till the imaging was done. Light sheet fluorescence microscopy (LFMS) was performed using UltraMicroscope II (LaVison BioTec, Germany). The samples were fixed with and immersed in absolute ECi solution in the mounting chamber during the imaging. Dual side illumination was used, and each plane was obtained every 7µm. Imaris software (Bitplane, Switzerland) was used to analyze the wholeorgan 3D images. The cell counts, volume and area of the granulomas were measured.

# Statistical Analysis

The cell count data were described as either mean ± standard deviation (flow cytometric studies) or mean ± standard error of the mean (histopathological and immunofluorescence studies, and tissue clearing). Prism version 7 (GraphPad Software, USA) was used for statistics and Student's t-test was used for analysis.

# RESULTS

Flow cytometric studies were performed in order to quantify three types of immune cells: neutrophils, alveolar macrophages, and infiltrating macrophages, in both naïve and Mtb-infected mice lungs (**Figure 1**). All the Mtb-infected mice used in this study were in their fourth week post-infection, at which point the total CFU of the Mtb (T = 4 weeks) showed a higher increase than that at the first day of infection (T = 1 day) (**Figure 1A**). As described in **Figure 1B**, flow cytometric studies of both naïve and Mtb-infected mice lungs were performed to compare the population of neutrophils (CD45+ and Ly6G+), alveolar macrophages (CD45+ and Siglec-F+), and infiltrating macrophages (CD45+ and CD64+) between two groups. The proportion of the immune cells showed a notable difference between two groups (**Figure 1C**). In the naïve group, resident alveolar macrophages (CD45+ and Siglec-F +) was the predominant cell type (3.97%). Neutrophils and infiltrating macrophages represented 2.74% and 1.77% of the cell population, respectively. In contrast, neutrophils were the most abundant cell type in Mtb-infected lung (8.10%). This was followed by infiltrating macrophages (4.26%), while the proportion of alveolar macrophages was relatively insignificant

(1.37%). The actual cell count of alveolar macrophages, however, showed no difference between two groups (**Figure 1D**). Instead the total cell number in the infected group was larger, making the relative proportion of alveolar macrophages smaller. Both neutrophils and infiltrating macrophages were drastically increased in the infected lung. The mean number of neutrophils was nearly 7 times larger than that of the uninfected lungs, and the mean number of infiltrating macrophages was more than 5 times larger than that of the uninfected lung. These data suggest that both neutrophils and macrophages mediate the host immune reaction against Mtb infection.

The morphology and the immune cell population were compared between the naïve and Mtb-infected mice lungs (**Figure 2**). At low magnification, the whole lung section could be viewed (**Figure 2A**, upper panel). The granulomas were well-demarcated from the surrounding tissue. At higher magnification, the granulomas showed a highly cellular lesion lacking the normal alveolar structure seen in the naïve lung (**Figure 2A**). This is consistent with a previous finding that granulomas in C57BL/6 mouse are exclusively cellular without any necrosis or hypoxia (Medina and North, 1998). In order to classify the types of cells in this highly cellular structure, immunofluorescence (IF) microscopy was performed (**Figure 2B**). Neutrophils, macrophages, and T lymphocytes were distinguished and counted. Since the lung samples were not perfused, most of the immune cells in the naïve mice were located within the alveolar vessels and the nongranulomatous area of the infected mice showed a similar distribution. In contrast, both neutrophils and macrophages were dispersed irregularly throughout the granuloma. The cell number of each subtype of immune cells was counted (**Figure 2C**). A slight difference in the immune cell population was observed between the granulomas and naive mice lungs, but the difference was not significant (**Figure 2C**). However, compared with the non-granulomatous area, the infected group contained a larger number of all three subtypes of immune cells than the naïve group (**Figure 2C**). As stated above, both H&E and immunofluorescence images reveal a limited view of the organ, as they are 2D sectioned images. The sectioned view represents only a part of the whole lesion, making it challenging to quantitatively compare normal and pathologic conditions.

To overcome the limit of 2D analytical methods, 3D wholelung imaging was performed in each of the cleared samples (**Figure 3**). In order to observe macrophages, the fluorescence was obtained using CX3CR1-GFP mice that produce green fluorescence protein (GFP) largely in macrophages (Jung et al., 2000) (**Figure 3A** and **Supplementary Videos 1**, **2**). In order to visualize neutrophils, LysM-GFP mice that has GFP in their neutrophils were used (Faust et al., 2000) (**Figure 3B** and **Supplementary Videos 3**, **4**). As the lung tissue itself has an auto-fluorescence (Ramanujam, 2000), the structures of the lungs such as the pleura or the bronchus could be identified along with the fluorescent cells (**Figures 3A,B** and **Supplementary Videos 1**–**4**). Multiple granulomas, whose intact shape and size were preserved, were detected in the infected lungs and their distribution in the entirety of the

lungs could be visualized (**Figures 3A,B**). Both neutrophils and macrophages were aggregated near the granulomas. Using zoomed-in images, a single granuloma could be selected and reconstructed into a graphic mass (green), whose volume could be measured (**Figure 3C** and **Supplementary Video 5**). Since the fluorescence of the background tissues could be deleted by modulating the field of intensity, the cellular components could be separately selected. The cells were marked as spots (green; **Figure 3C**). We analyzed the cell counts, specifically those within the borders of the granuloma in the infected samples and divided it by the volume of each granuloma. The number of macrophages were significantly higher in

the Mtb-infected granuloma (**Figure 3D**). In addition, the number of neutrophils were higher in the infected group than the naïve group (**Figure 3E**). These results were similar to that obtained by the flow cytometric studies. However, since the cell count was limited to the granulomas, this result is more reliable in regard to the analysis of the granulomatous lesions.

To map the vascular distribution of the organ, florescentlabeled L. esculentum lectin was infused via the retro-orbital vein to visualize blood vessels prior to sample preparation. It has been reported that uncontrolled angiogenesis occurs in the lungs following Mtb infection (Cronan et al., 2015; Oehlers et al., 2015). Consistent with this report, the vascular distribution differed between the naïve and Mtb-infected mouse's lungs (**Figure 3**). In the naïve mouse's lung, the vasculature was distributed evenly over the entire organ, showing typical alveolar structures (**Figures 3A,B**, upper right panels). However, in the Mtb-infected lungs, the vessels showed a more concentrated distribution around the granulomas (**Figures 3A,B**, lower right panels). In a zoomed-in image of a granuloma (**Figure 3C**), the vessels appeared to form an outer layer of the lesion, which suggests that the angiogenesis around the granulomas can cause a difference in vascular distribution.

## DISCUSSION

In this study, we employed a tissue-clearing method using ECi solution that preserves the fluorescence of fluorescent proteins, such as GFP, while making an intact organ transparent (Klingberg et al., 2017). Through this procedure, we were able to generate 3D images of the whole mouse lungs containing granulomatous lesions. We demonstrated the distribution of immune cells within each of the selected granulomas and performed a quantitative analysis comparing the immune cell population between Mtbinfected and naïve mouse lungs. The novelty of this study lies in its innovative approach to visualize the immune reactions during TB infection. Compared to the conventional methods such as H&E staining, FACS (fluorescence-activated cell sorting), clearing of the whole lung has a clear advantage of visualizing how the immune cells with fluorescence are located within the pathologic lesion. This allows an accurate counting of specific subtypes of immune cells, which is limited using 2Dsectioned images. In addition, since lung clearing uses the whole lung sample, macroscopic analysis, e.g., shape, volume, distribution of granuloma, can be performed simultaneously with the microscopic analysis. This method can be used in a variety of studies aiming to interpret or modulate the immune response against Mtb infection and should be further evaluated for its application in studying other diseases. For 3D images for the tissue-cleared lungs captured by light sheet microscopy, we employed low magnitude objective lens to acquire the larger area imaging including multiple granulomas, which may result in low-resolution images. To avoid a bias of manual counting of cell numbers from the image data, we performed cell counting by the Imaris software depending on the size and the intensity of the fluorescent cells. Although we just used only 4 weeks period of TB infection for immune cells counting in this study, as an innovative modality to visualize and quantify the immune reactions in TB infection, the whole lung clearing could be a versatile tool to find better clue for therapeutics against TB in combination with other experimental techniques.

#### DATA AVAILABILITY STATEMENT

All datasets generated for this study are included in the article/**Supplementary Material**.

#### ETHICS STATEMENT

The animal study was reviewed and approved by Yonsei University College of Medicine.

#### REFERENCES

# AUTHOR CONTRIBUTIONS

G-YK and H-JR performed the experiments and wrote the manuscript. H-HC provided essential reagents for the experiments. SS and Y-MH conceived the study and wrote the manuscript.

#### FUNDING

This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (NRF-2019R1A2C2003204, SS and NRF-2019R1A2C2008481, Y-MH) Republic of Korea.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcimb. 2020.00226/full#supplementary-material

Supplementary Video 1 | 3D clearing imaging of naïve CX3CR1-GFP+/− mouse's lung. Green and red fluorescence represents CX3CR1+/− cells and blood vessels, respectively.

Supplementary Video 2 | 3D clearing imaging of Mtb-infected CX3CR1-GFP+/− mouse's lung. Green and red fluorescence represents CX3CR1+/− cells and blood vessels, respectively. Blood vessels are aggregated around the granulomas.

Supplementary Video 3 | 3D clearing imaging of naïve LysM-GFP+/− mouse's lung. Green and red fluorescence represents LysM-GFP+/− cells and blood vessels, respectively.

Supplementary Video 4 | 3D view of Mtb-infected LysM-GFP+/− mouse's lung. Green and red fluorescence represents LysM-GFP+/− cells and blood vessels, respectively. Blood vessels are clumped around the granuloma lesion.

Supplementary Video 5 | A single granuloma of Mtb-infected LysM-GFP+/− mouse's lung. For analysis, a single granulomatous area was reconstructed into a graphic mass, and volume could be measured, also LysM-GFP+/− cells could be defined as green dots.

in the airways of patients with active pulmonary TB. Chest 137, 122–128. doi: 10.1378/chest.09-0903

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

Copyright © 2020 Kang, Rhyu, Choi, Shin and Hyun. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) 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.

# Myeloid C-Type Lectin Receptors in Tuberculosis and HIV Immunity: Insights Into Co-infection?

Kubra F. Naqvi and Janice J. Endsley\*

*Department of Microbiology and Immunology, The University of Texas Medical Branch, Galveston, TX, United States*

C-type lectin receptors (CLRs) are carbohydrate binding pattern recognition receptors (PRRs) which play a central role in host recognition of pathogenic microorganisms. Signaling through CLRs displayed on antigen presenting cells dictates important innate and adaptive immune responses. Several pathogens have evolved mechanisms to exploit the receptors or signaling pathways of the CLR system to gain entry or propagate in host cells. CLR responses to high priority pathogens such as *Mycobacterium tuberculosis (Mtb),* HIV, Ebola, and others are described and considered potential avenues for therapeutic intervention. *Mtb* and HIV are the leading causes of death due to infectious disease and have a synergistic relationship that further promotes aggressive disease in co-infected persons. Immune recognition through CLRs and other PRRs are important determinants of disease outcomes for both TB and HIV. Investigations of CLR responses to *Mtb* and HIV, to date, have primarily focused on single infection outcomes and do not account for the potential effects of co-infection. This review will focus on CLRs recognition of *Mtb* and HIV motifs. We will describe their respective roles in protective immunity and immune evasion or exploitation, as well as their potential as genetic determinants of disease susceptibility, and as avenues for development of therapeutic interventions. The potential convergence of CLR-driven responses of the innate and adaptive immune systems in the setting of *Mtb* and HIV co-infection will further be discussed relevant to disease pathogenesis and development of clinical interventions.

#### Edited by:

*Alexander S. Apt, Central Tuberculosis Research Institute (RAMS), Russia*

#### Reviewed by:

*Supriya Shukla, Case Western Reserve University, United States Konstantin Neumann, Hannover Medical School, Germany*

> \*Correspondence: *Janice J. Endsley<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Microbes and Innate Immunity, a section of the journal Frontiers in Cellular and Infection Microbiology*

> Received: *07 January 2020* Accepted: *04 May 2020* Published: *03 June 2020*

#### Citation:

*Naqvi KF and Endsley JJ (2020) Myeloid C-Type Lectin Receptors in Tuberculosis and HIV Immunity: Insights Into Co-infection? Front. Cell. Infect. Microbiol. 10:263. doi: 10.3389/fcimb.2020.00263* Keywords: tuberculosis, HIV, TB and HIV co-infection, innate immunity, C-type lectin receptors

# INTRODUCTION

TB and HIV are the leading causes of death by infectious agents globally (WHO, 2019a,b). In 2018, an estimated 10 million people fell ill with TB and 1.2 million deaths occurred among HIVnegative people globally (WHO, 2019a). Approximately 37.9 million people are currently living with HIV/AIDS and 1.1 million die each year (WHO, 2019b). Tuberculosis remains a large risk factor for people living with HIV (PLWH)/AIDS and HIV-associated TB was the cause of an additional 251,000 deaths in 2018. The co-infection of Mycobacterium tuberculosis (Mtb) and HIV contributes to the large burden on healthcare systems of endemic areas and have increased the priority of improved co-infection therapeutic strategies (WHO, 2019a,b).

Cell mediated immunity (CMI), especially that mediated by CD4+T cells, is essential for host resistance to Mtb (Cooper, 2009). HIV infection drives progressive depletion and dysfunction of leukocytes of the CMI response (Pawlowski et al., 2012) including defects that persist following antiretroviral therapy (ART). A synergistic deterioration of the immune system is associated with aggressive disease in those with co-infection through mechanisms that are incompletely characterized. Although the loss of protective immunity in PLWH is primarily attributed to CD4+T cell loss and dysfunction in the progression to AIDS, those with co-infection nonetheless display immune disturbance prior to significant T cell loss (Sharma et al., 2005; de Noronha et al., 2008; Sester et al., 2010).

Innate and adaptive immune cells which are resistant to direct infection display functional defects in the setting of HIV infection due to indirect effects on cell toxicity and immune signaling networks by viral mediators (Mazzuca et al., 2016; Garg and Joshi, 2017). Myeloid cell populations including monocytes, macrophages and dendritic cells are targets for HIV infection; macrophages in particular are an important viral reservoir (Igarashi et al., 2001; Heesters et al., 2015; Honeycutt et al., 2017). As the primary hosts for Mtb propagation, the direct and indirect effects of HIV infection on myeloid cell innate function are an important and poorly understood factor for the outcome of co-infection.

Signaling through myeloid cell PRRs dictates important innate recognition and responses to Mtb and HIV molecular patterns that may direct the progression of disease in co-infection scenarios. Engagement of PRRs such as toll-like receptors (TLR), nod-like receptors (NLR) and CLRs, and the downstream immune responses that are elicited is critical for dictating outcomes of individual disease during TB or HIV as previously reviewed (Mesman and Geijtenbeek, 2012; Hossain and Norazmi, 2013; Mortaz et al., 2015). In brief, TLRs interact with specific mycobacterial ligands to initiate phagosome maturation, proinflammatory, and anti-inflammatory cytokine secretion (e.g., IL-12, IL-18, and IL-10) (Kim et al., 2019). The resulting induction of IFN-γ further activates antimicrobial pathways of infected macrophages (Hossain and Norazmi, 2013; Mortaz et al., 2015). HIV infection activates TLR signaling pathways (Meier et al., 2007; Lester et al., 2008) that contribute to host restriction through induction of antiviral interferons or activation of cytokines that promote viral transcription (Mesman and Geijtenbeek, 2012).

Nod-like receptors are cytosolic PRRs which recognize intracellular bacterial and viral pathogens to further mediate macrophage activation. NOD-1 and 2 signaling following Mtb infection facilitates mycobacterial survival by suppressing apoptosis of macrophages (Mortaz et al., 2015). Additionally, NLRP3-dependent inflammasome activation of macrophages exposed in vitro to Mtb promotes IL-1β secretion and cell death (Mortaz et al., 2015). Inflammasome activation by NOD-like receptors is also a major mechanism of pyroptotic CD4+T cell depletion due to HIV in ex vivo models of infection (Doitsh et al., 2014; Tomalka et al., 2016). HIV genomic RNA and newly synthesized mRNA in infected macrophages is also detected by the Rig-I like receptors (RLR) in the cytosol. RLRs drive the production of type-1 IFNs following recognition of intracellular pathogens (Bergantz et al., 2019), inducing expression of interferon stimulated genes and eliciting an antiviral response. However, HIV can evade this mechanism through degradation of RIG-I by a viral protease (Bergantz et al., 2019).

The surface bound CLRs, especially abundant on innate leukocytes of the myeloid lineage, also bind to molecular patterns of Mtb and HIV (Turville et al., 2003; Mishra et al., 2017). The characteristic glycosylation of the mycobacterial cell wall and HIV envelope protein gp120 (Turville et al., 2001; Guttman et al., 2015; Ishikawa et al., 2017), engage CLRs, which recognize carbohydrate motifs. Molecular recognition through CLRs triggers a myriad of downstream signaling events that modulate ligand and CLR-specific immune outcomes (Geijtenbeek and Gringhuis, 2009). Our understanding of the role of CLRs in TB and HIV mono-infections is rapidly evolving through active investigation. In contrast, the effect of co-infection to exacerbate, confound, or otherwise perturb signaling through CLR pathways is largely uncharacterized.

This review will discuss the current state of knowledge regarding CLRs with described roles in innate immune recognition of Mtb and HIV. The contribution of these CLR pathways to protective and non-protective immune outcomes will be discussed in the context of mono- and or co-infection settings. Finally, the potential to exploit CLR pathways for clinical interventions that prevent or reduce disease due to this important dual pandemic will be discussed.

#### CLRS REGULATE PROTECTIVE AND PATHOGENIC OUTCOMES OF TB AND HIV

Dendritic cells (DCs) and macrophages are sentinel antigen presenting cells (APC) and are therefore well equipped with PRRs to engage molecular motifs of Mtb or HIV during infection. The CLRs of antigen presenting cells bind self and non-self-antigens to activate and/or suppress immune function and internalize pathogens (McGreal et al., 2005). CLRs can be classified based on structural, carbohydrate recognition and signaling features (Dambuza and Brown, 2015). The carbohydrate recognition domain is a compact structural motif of CLRs which determines carbohydrate specificity and binds ligands in Ca2+ dependent or independent manner (Geijtenbeek and Gringhuis, 2009).

The transmembrane CLRs are further divided into groups, the type I mannose receptor family and the type II asialoglycoprotein receptor family which includes further subfamilies (dectin 1 and DCIR) (Geijtenbeek and Gringhuis, 2009). The signaling motifs of CLRs can also be used to categorize these receptors further. Transmembrane CLRs can include immunoreceptor tyrosine-based activation (ITAM) or inhibitory (ITIM) motifs while some CLRs signal through a single tyrosine-based motif termed hem-ITAM (Osorio and Reis e Sousa, 2011). Many transmembrane and soluble CLRs carry out essential functions in immune homeostasis and innate responses against a wide variety of pathogens. This review will focus on CLRs associated with Mtb and HIV infections including the mannose receptor, Mincle, Dectins 1 and 2, Mannose Binding Lectin, DC-SIGN, Langerin and DCIR (**Figure 1**).

respectively. As another mechanism of host defense, the macrophage mannose receptor restricts viral particle budding, although HIV can evade this mechanism by downregulating the *mrc1* gene. HIV also increases Mincle-induced pro-inflammatory cytokine production in *in vitro* models of infection. Together, these CLRs

demonstrate the multifaceted role of receptor binding in HIV protection and pathogenesis and regulating the innate immune response to *Mtb*.

#### MANNOSE RECEPTOR

The mannose receptor (MR, CD206) is a type I transmembrane CLR present on alveolar macrophages, M2 macrophages and dendritic cells. Binding of MR to sulfated and mannosylated sugars results in internalization of antigen and delivery to the MHC II and CD1b pathways (Martinez-Pomares, 2012). Receptor engagement by Mtb has served as a model system to characterize MR signaling outcomes and further define an innate immune role in TB. Virulent Mtb induces PPAR-γ-dependent inflammatory responses, including IL-8 and cyclooxygenase 2 expression, through a MR dependent pathway (Rajaram et al., 2010). MR binds to Mtb liparabinomannan and mannosylated proteins, resulting in interaction with the FcRγ-chain which mediates surface localization and activation of the Grb2 adaptor molecule required for phagocytosis (Rajaram et al., 2017). MR recruitment of the tyrosine phosphatase, SHP-1, however, limits the PI(3)P production needed for maturation of the Mtbcontaining phagosome (Rajaram et al., 2017). As currently understood, recognition of Mtb PAMPs through MR activates important inflammatory and phagocytic function of APCs, but may also promote intracellular Mtb survival through inhibition of phagosome-lysosome fusion.

The distribution of MR as a phenotypic marker of alternatively activated, or M2, macrophages in unique subsets and disease processes has also informed our understanding of its immune role. MR is a selective marker of perivascular macrophages in human and non-human primate CNS tissue and a marker of phenotypic switch (CD206+ to CD206-) during HIV neuroinflammation (Holder et al., 2014). Expression of MR on peripheral macrophages can also serve as a potential biomarker to identify those with dual HIV strain infections. MR expression decreases following ART treatment for patients with HIV-1 or HIV-2, and remains constant in those with dual infection despite treatment (Andersen et al., 2018). Cellular expression of MR also contributes to sexual transmission of HIV as an alternative entry receptor in vaginal epithelial cells, the primary site of male-to-female transmission (Jadhav et al., 2013).

Binding of MR to HIV gp120 induces production of matrix metalloproteinase (MMPs) breaking down the extracellular matrix and permitting HIV to cross the epithelial barrier to gain access to sub-epithelial CD4+ cells (Fanibunda et al., 2011). In addition to expression on vaginal epithelial cells, MR expression on sperm cells has been shown in vitro to mediate infection to susceptible cells, suggesting a possible role for sperm cells as vectors for sexual transmission of HIV (Fanibunda et al., 2008; Cardona-Maya et al., 2011). Binding of HIV gp120 to MR is further enhanced by oligomerization of the receptor on macrophages and dendritic cells, further promoting transinfection of CD4+T lymphocytes (Lai et al., 2009). An antiviral role for MR has also been described and occurs through prevention of HIV particle release from infected cells (Sukegawa et al., 2018). Interestingly, HIV counteracts this antiviral activity by repressing MR transcription (Sukegawa et al., 2018).

The role for MR in the innate function of APCs in response to Mtb or HIV is increasingly understood to be complex; activating important antimicrobial mechanisms and serving as an avenue for immune exploitation. To date, studies in the setting of experimental co-infection that would show how simultaneous or synergistic activation through MR signaling could impact myeloid cell function are lacking. These are important given the potential for co-infection signaling events to influence disease processes. Speculatively, the Mtb-driven MR signaling of inflammation could augment viral replication driven through cytokine pathways known to activate the HIV promoter (Jiang and Dandekar, 2015). Similarly, activation of MMPs by HIV signaling through MR could affect the development and maintenance of Mtb granulomas which are important for mycobacterial containment (Al Shammari et al., 2015; Sabir et al., 2019).

#### MINCLE

Macrophage-inducible C-type lectin (Mincle, Clec4e), is a type II transmembrane CLR expressed by myeloid cells, such as macrophages, and contains a single carbohydrate recognition domain. Mincle binds both foreign and self-antigens including motifs found on fungal and bacterial surfaces and damagedself antigens including SAP130 and β-glucosylceramide that are released from necrotic cells (Miyake et al., 2010; Nagata et al., 2017). Binding of Mincle to fungal pathogens such as C. albicans and Malassezia species is an important component of anti-fungal immunity mediated through TNF-α induction. The binding of Mincle to trehalose-6,6'-dimycolate (TDM) of the mycobacterial membrane activates macrophages to produce nitric oxide(NO) and inflammatory cytokines that contribute to antibacterial function and granuloma formation, respectively (Miyake et al., 2010). Consistent with other CLRs, Mincle deficiency does not alter the disease outcome in experimental murine TB. The inflammatory response that occurs upon in vitro binding of Mincle by TDM (Lang, 2013) is also fully compensated for in vivo (Heitmann et al., 2013).

In contrast to Mincle, the macrophage C-type lectin (MCL) co-receptor for Mincle has been shown to be indispensable for protective TB immunity (Wilson et al., 2015). MCL−/− mice exhibit increased mortality, bacterial burden and pulmonary inflammation following Mtb infection, compared to WT. MCL shares high AA sequence homology with Mincle and is also expressed by myeloid populations (Miyake et al., 2013, 2015). Mincle expression is positively regulated by MCL, an outcome postulated to reflect the heteromeric complex formation of Mincle and MCL (Miyake et al., 2015). Formation of the Mincle/MCL complex occurs downstream of Mincledependent MyD88 signaling and contributes to protective antimycobacterial responses (Kerscher et al., 2016).

A role for Mincle in HIV disease outcomes has not been described to date. Similarly, the contribution of Mincle signaling to co-infection outcomes is poorly understood due to a lack of knowledge related to HIV activation and paucity of investigations of experimental co-infections. A recent study, however, suggested that peripheral blood myeloid cells from HIV+ subjects respond differently to Mtb Mincle agonists (Zapata et al., 2019). Interestingly, in vitro activation of Mincle using a TDM analog promoted increased pro-inflammatory responses by peripheral blood monocytes from HIV+ and aged subjects (Zapata et al., 2019). These results suggest that HIV infection may disturb Mincle signaling in the setting of co-infection with Mtb and potentially alter immune outcomes.

# DECTIN 1

The type II transmembrane CLR, Dectin-1, is expressed on myeloid cells including monocytes, macrophages, dendritic cells and neutrophils, and binds to β-glucans commonly found on fungal cell walls (Tsoni and Brown, 2008). Ligand binding of Dectin-1 induces a downstream signaling response which results in phagocytosis, respiratory burst, and a pro-inflammatory response that promotes fungal clearance (Brown, 2006; Dennehy and Brown, 2007). In addition to regulating innate immune responses to pathogens, Dectin-1-induced cytokines modulate subsequent adaptive responses (Brown, 2006). Blocking Dectin-1 signaling in a human PBMC model of in vitro Mtb infection reduced production of IL-17A through a mechanism that required co-signaling with TLR4 and endogenous IL-1 (van de Veerdonk et al., 2010). The IL-17 producing helper T cells (Th17) are among the essential CD4+T cell subsets that direct anti-mycobacterial responses of the adaptive immune system. The Dectin-1 pathway could thus be exploited as a novel vaccination strategy to activate Th17 cell memory to Mtb and other important pathogens that rely on IL-17 for protective immunity (Scriba et al., 2008).

Similar to Mincle, the immune functions activated through Dectin-1 appear to have redundant mechanisms, as Dectin 1 deficient mice display similar immune response and pulmonary disease as wild type counterparts (Marakalala et al., 2011). Deficiency of a single signaling intermediate rarely results in markedly increased pulmonary TB. An exception is deficiency of CARD9, the central adaptor for many CLRs including both Mincle and Dectin-1. Mtb-infected mice lacking CARD9 display severe pyogenic and neutrophilic pneumonia, and fatal TB disease (Dorhoi et al., 2010). Interestingly, deficiency in CARD9 did not impair the in vitro antimicrobial activity of macrophages and did not alter Th17 or other T cell responses in vivo (Dorhoi et al., 2010). The substantial influx of neutrophils observed in Card9−/− mice is consistent with observations that loss of signaling through MCL or CTLR (Clec9a) compromises the regulation of IL-1 and CXCL8 responses of Mtb-infected macrophages (Wilson et al., 2015; Cheng et al., 2017).

In contrast to the activation of protective immune responses to Mtb, HIV engagement of Dectin-1 promotes viral replication by facilitating cis-infection of immature DCs (Cote et al., 2013). Dectin-1 engagement by opportunistic pathogens can also promote viral pathogenesis. Binding of Dectin-1 by β-glucans of fungal pathogens such as histoplasmosis results in reduced leukotriene B4 levels (Sorgi et al., 2009). An imbalance between leukotrienes and prostaglandins favors HIV proliferation and establishment of opportunistic infection due to the role of PGE2 in inhibiting microbial killing (Sorgi et al., 2009). The balance between leukotrienes and prostaglandins also influences survival in experimental models of TB and are identified as a correlate of TB-diabetes co-morbidity (Tobin et al., 2012; Mayer-Barber et al., 2014; Shivakoti et al., 2019).

## DECTIN-2

Dectin-2, present on tissue macrophages, some DC subsets including Langerhans cells, and peripheral blood monocytes, recognizes numerous fungal and bacterial pathogens (Graham and Brown, 2009). Dectin-2 binds zymosan of fungal species such as Candida albicans and mannose rich glycolipids such as lipoarabinomannan (Man-LAM) of Mtb (Decout et al., 2018). Binding of Dectin-2 to Man-LAM activates pro-inflammatory (IL-6 and TNF-α) and immune regulatory (IL-10 and TGF- β) cytokines (Yonekawa et al., 2014). Consistent with the roles of IL-6 and TGF- β to promote Th17 differentiation, binding of Man-LAM by Dectin-2 drives generation of Mtb-specific Th17 cells (Decout et al., 2018).

Although other Dectin-2 family CLRs, such as DCIR, are associated with HIV transmission, a role for Dectin-2 signaling in HIV mono-infection and co-infections with Mtb has not been described. The function of both Dectin 1 and 2 to promote differentiation of Th17 cells during the adaptive response to other pathogens such as Mtb, however, may have consequences in the setting of HIV co-infection. Activated CD4+T cells with a memory phenotype are more susceptible to HIV infection and support greater viral replication than resting T cell counterparts. Among various CD4+T cell populations, the Th17 populations are especially susceptible to HIV infection, lacking inhibitory RNAses that limit viral replication in other cells (Sun et al., 2015; Christensen-Quick et al., 2016; Lee and Lichterfeld, 2016; Fernandes et al., 2017). Th17 cells expressing CCR6 were recently described to function as a long lived HIV reservoir that persists in PLWH despite ART pressure (Sun et al., 2015; Fernandes et al., 2017; Gosselin et al., 2017). CLR-dependent pathways that promote differentiation of Mtb-specific Th17 cells may thus contribute to HIV proliferation and persistence. Depletion and compromise of Th17 cells due to HIV infection could similarly compromise protective function of the cell mediated immune response to Mtb.

## MANNOSE BINDING LECTIN

Mannose binding lectin (MBL) belongs to a family of secreted proteins known as collectins and primarily circulates through serum, synovial and amniotic fluid. MBL ligand binding is Ca2+ dependent and selective for terminal mannose, fucose and N-acetylgalactosamine (GalNAC)(Ip et al., 2009). Due to the broad ligand binding and presence in the serum, MBL is an important receptor for opsonization of pathogens to clear infection and prevent recurrent infections (Ip et al., 2009). MBL binding to Mycobacterium avium was previously reported (Polotsky et al., 1997), while recent studies have demonstrated a broader role in mycobacterial immunity. Ligation of MBL to Mtb and the related M. bovis, results in enhanced phagocytosis, lectin pathway activation and agglutination of bacteria (Bartlomiejczyk et al., 2014). Additionally, binding of MBL to Mtb activates MASP1 homodimers to catalyze activation of MASP2 and ultimately generation of C3 convertase (Klassert et al., 2018) in the immune complement cascade. The function and expression of MBL is dependent on polymorphisms in the MBL2 gene, which encodes the receptor. Polymorphisms in MBL2 are associated with susceptibility to pulmonary and extra-pulmonary TB in various human populations (da Cruz et al., 2013; Nisihara et al., 2018).

The HIV envelope displays several conserved glycan residues that are epitopes for broadly neutralizing antibodies (bnAb) produced by B cells of the acquired immune system. MBL present in human serum is able to block binding of the 2G12 bnAb to HIV, and as a result, increase virus infectivity (Marzi et al., 2007). Serum concentrations of MBL may thus limit the efficacy of neutralizing antibody-based prevention or treatment strategies for HIV. The outcome of MBL signaling in the setting of co-infection has not been described to date. Activation of MBL by Mtb exposure could be explored as a potential mechanism for the increased viral replication observed in those with dual infection. Similarly, increased HIV replication due to inhibition of bnAb by MBL could further compromise CMI to Mtb.

# DC-SIGN

The CLR which has been most extensively studied in Mtb and HIV infections is DC-SIGN. This mannose-binding type II CLR is present on dermal DCs, interstitial DCs of the mucosa, and macrophages (van Kooyk and Geijtenbeek, 2003). DC-SIGN interacts with the LPS structure of Klebsiella pneumonia and Helicobacter pylori to internalize and target these pathogens for antigen presentation (van Kooyk and Geijtenbeek, 2003). In experimental TB, selective knockout of the murine homolog for human DC-SIGN (Tanne and Neyrolles, 2010), SIGNR1, only partially impairs host resistance to Mtb. However, deficiency in both SIGNR1 and murine MR results in increased lung inflammation and larger foci of bacilli 5 months post-infection (Court et al., 2010). These disease outcomes are moderate, however, compared to those in mice lacking the essential cytokine pathways (TNF, IL-1 or IFN-γ) which lack compensatory mechanisms and are unable to control Mtb (Court et al., 2010).

Although DC-SIGN is primarily expressed on dendritic cells, expression on immunoregulatory macrophages has been shown in the pleural cavity of human patients and lung tissue from NHPs with active TB (Lugo-Villarino et al., 2018). The functional outcome of DC-SIGN signaling within Mtb-exposed myeloid cells remains to be fully characterized, however, modulation of the Dectin-1 driven pro-inflammatory responses has been observed in a subset of M2 macrophages (Lugo-Villarino et al., 2018). Further investigations are required to determine if DC-SIGN pathways may work in concert with other CLRs or PRRs to regulate damaging pro-inflammatory responses to Mtb.

DC-SIGN also serves as an entry receptor for several viral pathogens including Dengue virus, Ebola virus, hepatitis C virus and cytomegalovirus (van Kooyk and Geijtenbeek, 2003). A role for DC-SIGN in trans-infection with HIV and the closely related simian immunodeficiency virus (SIV) has been well-documented in human and non-human primate cells, respectively (Geijtenbeek et al., 2001). Adhesion of gp120 to DC-SIGN promotes viral transmission via the lymphatics while expression of DC-SIGN by placental Hofbauer cells permits vertical transmission of HIV (Soilleux et al., 2001; da Silva R. C. et al., 2011). Expression of DC-SIGN has also been described on HIV-susceptible tissues such as colonic mucosa (Preza et al., 2014) and foreskin epithelium (Hirbod et al., 2010) as a potential mechanism of HIV transmission to and from those sites.

The function of DC-SIGN in viral infection and transmission highlights the specificity and overlap of the CLR interactions with HIV. Although DC-SIGN promotes trans-infection of HIV, many other CLRs carry out a similar function independent of DC-SIGN. As previously discussed, MR facilitates HIV transmission by binding to mannose moieties of gp120 through a mechanism inhibited by pre-incubation of cells with mannan (Nguyen and Hildreth, 2003). The heavy glycosylation of HIV gp120 also contains sialic acid residues which are recognized by siglecs. These sialic acid binding lectins, present on macrophages and DCs, facilitate viral adhesion and infection of R5-tropic viruses to macrophages (Zou et al., 2011). In particular, siglec-1 expressed on mature DCs of the cervical mucosa enhances viral uptake and trans-infection, thus enabling systemic viral spread (Izquierdo-Useros et al., 2014; Perez-Zsolt et al., 2019).

Soluble CLRs, such as galectin-1, are exploited by HIV to enhance binding of gp120 and the CD4 receptor to facilitate viral infection of T cells (St-Pierre et al., 2011). CLRs which bind to HIV gp120 are also able to competitively inhibit viral binding to DC-SIGN and thus, block trans-infection. The CLR surfactant protein SP-D, present in mucosal tissue, inhibits viral entry and DC-SIGN interaction by competitively binding HIV gp120 (Dodagatta-Marri et al., 2017). In short, CLR binding to gp120 and other carbohydrate rich molecular motifs is an important mechanism for HIV pathogenesis as well as a potential avenue for clinical intervention.

The association of DC-SIGN in immune evasion strategies employed by Mtb and HIV has been characterized as previously reviewed (Kaufmann and Schaible, 2003; van Kooyk et al., 2003). In brief, during co-infection with Mtb, DC-SIGN mediated Raf-1 signaling has been shown to be critical for HIV transcription elongation and can even enhance viral replication (Gringhuis et al., 2010). Additional studies of the antigen uptake mechanism of DC-SIGN have identified the involvement of membrane cholesterol and dynamin for endocytosis and the clatherin-dependent HIV internalization into dendritic cells (Cambi et al., 2009).

# LANGERIN

The CLR Langerin is specifically expressed by Langerhans immature DC of the epidermis and mucosal tissues (de Witte et al., 2008). Langerin is a type II transmembrane receptor which binds mannose, fucose, and GlcNAC in a Ca2+ dependent manner (van der Vlist and Geijtenbeek, 2010). As a consequence of broad spectrum carbohydrate recognition, Langerin recognizes a number of PAMPs including those expressed by Candida albicans and Mycobacterium leprae. To date, the subsequent downstream signaling events triggered by Langerin binding are poorly characterized. Additionally, although it is likely that Langerin recognizes components of the glycosylated Mtb cell wall, direct binding has not been described (van der Vlist and Geijtenbeek, 2010).

Langerhans cells are among the first innate immune cells to encounter mucosal pathogens and are therefore important gatekeepers to prevent infection at those sites (van der Vlist and Geijtenbeek, 2010). To that effect, binding of Langerin to HIV initiates a TRIM5α dependent pathway which targets HIV for autophagic degradation (Ribeiro et al., 2016). Interestingly, DC-SIGN engagement abrogates this restriction pathway by causing a dissociation of TRIM5α from the receptor and thus, permitting infection (Ribeiro et al., 2016). These findings highlight the synergistic ability of DC CLRs to dictate the outcome of HIV infection by permitting or restricting pathogen replication and immune responses. Additionally, therapeutic strategies which block DC-SIGN may in turn augment the protective function of Langerin signaling during HIV infection. Identification of Mtb binding by Langerin could suggest avenues whereby Langerin and DC-SIGN signaling convergence, or interference, may impact immune outcomes in co-infected individuals.

#### DCIR

Dendritic cell immunoreceptor (DCIR) is highly expressed on peripheral blood leukocytes including monocyte-derived DCs, macrophages, neutrophils, and plasmacytoid DCs (Graham and Brown, 2009). DCIR contains an immunoreceptor tyrosinebased inhibition motif (ITIM) associated with downregulation of pro-inflammatory signaling. Similar to other related CLRs, DCIR-dependent internalization targets antigens for presentation on APCs, thus promoting T-cell proliferation (Graham and Brown, 2009). As mentioned previously, the DCIR related CLR, Dectin-2, promotes Th17 responses upon engagement with Mtb antigen (Decout et al., 2018). Similarly, recognition of Mtb by DCIR promotes Th17 T cell differentiation while also activating the IFNR-associated JAK-STAT pathway to sustain type I IFN signaling and reduce Th1 responses. Expectedly, knockout of DCIR improves Mtb control but also results in increased pulmonary pathology (Troegeler et al., 2017). More interesting, however, is the immune activating nature of DCIR in TB immunity which is unexpected for an ITIM-containing CLR, although this mechanism is yet to be determined (Troegeler et al., 2017).

Analogous to DC-SIGN, DCIR plays an important role in promoting HIV viral capture and replication within CD4+T cells through activating phosphorylation of the receptor ITIM domain (Lambert et al., 2008, 2011). Blocking of DCIR has been employed as a therapeutic strategy to prevent HIV transmission and may be an effective strategy to improve Th1 responses against Mtb in a co-infection scenario. The role of DCIR to promote Th17 differentiation, similar to Dectin 1 and 2, may increase viral replication and persistence due to the susceptibility of Th17 cells to HIV.

### GENETIC POLYMORPHISMS OF CLRs AS DISEASE PREDICTORS

Polymorphisms of PRR genes within the human population have been increasingly associated with disease resistance and susceptibility. Functional polymorphisms in CLR genes that result in receptor impairment have been linked to increased susceptibility to infections such as invasive pneumonia and hepatitis (Eisen and Minchinton, 2003), as well as chronic diseases such as Crohn's disease (Marquez et al., 2009). The population diversity of several CLRs, especially MR, Mincle, and MBL, has been further linked to increased susceptibility to HIV and Mtb within endemic areas through diverse mechanisms including: modulation of immune outcomes, improved pathogen transmission, and associated co-morbidities.

Recognition of Man-LAM by MR plays a key role in innate immunity to Mtb (Kang et al., 2005). A single nucleotide polymorphism (SNP) of the MRC1 gene which encodes the MR was recently identified as a candidate for genetic susceptibility to Mtb. A cohort of 222 Chinese subjects with pulmonary TB was compared to 232 healthy controls and analyzed for six SNPs in exon 7 of the MRC1 gene. In both frequency of genotype and allele, the G1186A site was significantly different between those with pulmonary TB compared to healthy controls (Zhang et al., 2012). These observations identify the G1186A mutation as a potential risk factor for the development of pulmonary TB. Speculatively, functional changes in MR due to the G1186A SNP may temper the pro-inflammatory or phagocytic outcomes of MR signaling upon Mtb infection, or alternatively, further inhibit phagosome maturation. To date, MR SNP that are linked to susceptibility to HIV and HIV/Mtb co-infections have not been described.

Polymorphisms in the CLEC4E gene that encodes Mincle have been associated with susceptibility to Mtb, although limited results to date suggest racial or other population differences. Mincle binding of mycobacterial TDM is strongly associated with TB granuloma formation. Four CLEC4E SNPs (rs10841845, rs10841847, rs10841856, and rs4620776) were identified in an African patient cohort of 416 confirmed TB cases and 405 healthy controls (Bowker et al., 2016). No significant association between these four SNP and TB disease was identified in this comparison (Bowker et al., 2016). A more recent study in a population from northern China further characterized these CLEC4E polymorphisms in association with pulmonary TB disease (Kabuye et al., 2019). Interestingly, studies in this population identified significantly increased variants of rs10841845 and rs10841847 in the 214 control patients compared to the 202 pulmonary TB patients. This suggests that functional polymorphism of CLEC4E may confer protection against pulmonary Mtb infection in this population (Kabuye et al., 2019). To date, MRC1 and CLEC4E SNP that are linked to risk for HIV infection or HIV/Mtb co-infections have not been described.

Human MBL, encoded by the MBL2 gene, belongs to a family of soluble CLRs known as collectins that initiate complement activation. Polymorphisms of MBL2 and the downstream signaling components which initiate the complement lectin pathway (Klassert et al., 2018), are associated with susceptibility to both pulmonary and extra-pulmonary TB across various populations (Denholm et al., 2010). In a cohort from the northeastern region of Brazil, MBL2 promoter polymorphisms were linked with increased susceptibility to pulmonary TB. Interestingly, no significant correlations were observed between MBL2 polymorphisms and extra pulmonary TB (da Cruz et al., 2013). Conversely, a relationship with extra-pulmonary TB was identified in a cohort of Chinese patients. In this analysis, MBL2 polymorphisms were found to correlate with a diagnosis of spinal TB (Zheng et al., 2018).

MBL2 polymorphisms have also been associated with reduced levels of serum MBL, although the implications for Mtb susceptibility remain to be fully characterized (da Cruz et al., 2013). In one study of patients with ankylosing spondylitis (AS), a rheumatic disease characterized by low MBL levels, a significant correlation was identified between MBL deficiency and TB. The increased TB risk in those with low serum MBL was independent of either TNF therapy or latent infection in the patient medical history (Nisihara et al., 2018).

Susceptibility to HIV infection is also associated with MBL polymorphisms. In a 2011 study of a cohort from Southern Brazil, variant alleles of MBL were associated with increased susceptibility to HIV-1 infection (da Silva G. K. et al., 2011). Genotyping of HIV-infected patients of European and African descent in the cohort further revealed a difference in MBL2 SNP frequencies and serum levels between ethnic groups. Interestingly, HIV-positive patients of European descent had low MBL serum levels whereas those of African descent had elevated MBL levels. The basis for this dichotomy is unknown and suggests that the full scope of immune responses regulated by MBL2 are not yet understood.

In one report, MBL2 alleles and genotypes were assessed in a Western European population for association with coinfection. Interestingly, HIV/TB dual disease was associated with low or non-producer alleles in comparisons with TB negative subjects including those with a positive HIV status (Garcia-Laorden et al., 2006). MBL deficiency may thus be associated with active TB in HIV co-infection. These findings demonstrate the broad spectrum of MBL2 polymorphisms associated with HIV and TB disease across various populations, including those with co-infection.

# CLR BASED THERAPEUTIC STRATEGIES

The function of C-type lectin receptor signaling to shape innate and adaptive immune responses is exploited in recent approaches to develop novel therapeutics. CLR receptors are efficiently engaged by Mtb and HIV PAMPs and several of the resulting immune outcomes are characterized (**Figure 2**). As a result, mycobacterial and viral antigens that bind CLR have many therapeutic applications ranging from immune activation to cell targeted delivery. Competitive inhibition of CLR binding to viral particles also holds promise as a strategy for antiviral therapy.

#### Vaccine Adjuvants

The glycosylation of both the mycobacterial cell wall and HIV virion is an important factor for recognition by CLRs and initiates downstream immune responses. Engagement of Mincle by TDM (Ishikawa et al., 2009) and phosphatidylinositol mannosides (Mosaiab et al., 2018) of mycobacteria induces robust proinflammatory cytokine response (IL-6 and TNF-α) and NO production in vivo (Ishikawa et al., 2009). Activation of Mincle signaling using TDM derivatives has been employed as an adjuvant strategy for TB vaccines (Desel et al., 2013; Decout et al., 2017) as well as for vaccines for unrelated pathogens (Azuma and Seya, 2001; Drummond et al., 2011). Of note, the adjuvant effect of TDM is dependent not only on Mincle, but also its co-receptor, MCL, constitutively expressed on myeloid cells (Tanne and Neyrolles, 2010; Miyake et al., 2013). Adjuvant outcomes could thus be tailored as needed for specific immune responses through strategies that target individual or multiple CLR pathways.

The choice of antibody subclass can also be modulated through CLR signaling. Generation of balanced IgG2a/IgG1 antibody subclasses specific to HIV is an important goal of DNA immunization. Activation of CLR signaling by HIV envelope was shown to regulate the antibody subclass response, as demonstrated by immunization of Card9−/− mice (Hess et al., 2019). In contrast, antibody subclasses were not altered in Myd88/Trif −/− mice that lack functional TLR signaling (Hess et al., 2019). Application of CLR-targeting molecules for vaccine adjuvants is at an early stage of development. These early outcomes show promise to "fine-tune" the immune response as relevant to different microbial threats.

### Cell Targeted Delivery

Not only are CLRs capable of eliciting protective cytokine responses, but they also function as internalization receptors for delivery to antigen presentation pathways. Thus, the inclusion of CLR targets is increasingly employed to improve cellular uptake of drugs or vaccines. In a recent report, CLR-targeted delivery was shown to improve inhaled delivery of lipid nanoparticles loaded with rifampicin, a first line TB drug (Maretti et al., 2019). Decoration of lipid nanoparticles with novel mannose derivatives targeted the particle for uptake by MR. The result was improved efficiency of drug uptake by alveolar macrophages, the host cell for Mtb (Maretti et al., 2019).

A nanoparticle vaccine strategy that incorporated CLR targeted oligosaccharides has also been employed as a novel vaccination strategy for HIV. Co-delivery of mannose-enriched ligands that target DC-SIGN, with HIV peptides, increased the generation of antigen-specific CD8+ and CD4+T-cell responses (Climent et al., 2018). Targeting antigens to DCs through DC-SIGN has also been employed for TB vaccination. Conjugation of anti-DC-SIGN antibodies to the Mtb antigen Ag85B molecule elicited strong antigenspecific and polyfunctional (IFN-γ+, IL-2+, and TNF-α+) CD4+T cell responses (Velasquez et al., 2018). These strategies to target DC-SIGN may increase uptake and targeting to antigen presentation pathways as well as activate immune danger signals that promote development of polyfunctional T cell memory.

#### Receptor Antagonists

The function of CLRs as potent endocytic receptors and innate immune activators is frequently exploited by pathogens for transmission and pathogenesis. Inhibition of these interactions thus provides opportunities for therapeutic intervention. Recently, blocking CLR-mediated viral transmission to naïve host cells was shown to serve as a host-directed therapeutic strategy for HIV. Galectin-1, a soluble CLR used by HIV for attachment and entry, may also serve as a target for antiretroviral therapy. Treatment with carbohydrate lactoside compounds competitively inhibit the galectin-1 interaction with human CD4+T cells and HIV, and thus preventing attachment to uninfected cells (St-Pierre et al., 2012). Direct carbohydrate inhibitors are effective treatment strategies for CLRs with unique ligands. The carbohydrate recognition motifs shared by different CLRs are more challenging to target through inhibition strategies. For example, DC-SIGN shares ligand homology with Langerin. DC-SIGN is exploited by HIV to

facilitate viral transmission while Langerin directs protective outcomes (de Witte et al., 2008; van der Vlist and Geijtenbeek, 2010).

One potential strategy is the use of glycomimetic technology to synthesize receptor targeted inhibitors specific for individual CLRs. Use of a glycomimetic to inhibit DC-SIGN binding reduced HIV trans-infection of T cells in human explant tissue models (Varga et al., 2014). At low µM concentration, these functionalized multivalent ligands also inhibited in vitro HIV trans-infection in isolated CD4+T cells (Varga, Sutkeviciute et al.). Blocking of other receptors which facilitate trans-infection of HIV, such as DCIR, have also shown efficacy in decreasing viral infection of CD4+T cells (Lambert et al., 2011). Use of glycomimetics and other CLR inhibitors thus have potential to reduce infections and may also have applications to modulate disease outcomes.

#### CONCLUDING REMARKS

CLRs are essential PRR pathways of the innate immune response with diverse roles in immunity to Mtb and HIV. Our understanding of the roles of individual CLRs and the downstream immune responses in Mtb and HIV infection, and especially co-infection, is still evolving. Several CLRs bind Mtb ligands and activate innate, often pro-inflammatory, immune responses. There is redundancy in CLR function, as individual CLRs are generally dispensable with regards to TB disease outcomes in murine models. The roles for CLRs in HIV infection are less characterized due to the limitations for animal models. As currently understood, CLRs can generate protective anti-viral responses as well as serves as mechanisms for viral transmission. In the setting of co-infection, simultaneous signaling could drive convergent processes that differ from mono-infection and contribute to aggressive disease through mechanisms that are not yet understood. These overlapping mechanisms may also extend into other opportunistic infections and co-morbidities associated with Mtb or HIV infections, which may modulate innate immune signaling of CLRs. On-going applications that target CLR pathways demonstrate recognition of the translational potential for development of interventions for TB and HIV.

# AUTHOR CONTRIBUTIONS

KN and JE both contributed conception and design of the review. KN wrote the first draft of the manuscript and developed the figure graphics. JE wrote sections of the manuscript and provided overall editing. All authors contributed to manuscript revision, read and approved the submitted version.

# FUNDING

This work was supported by funding from NIH, NIAID to JE (R61AI138328). Fellowship support for KN was provided by a pre-doctoral award from the James W. McLaughlin Endowment.

# REFERENCES

tuberculosis infection in a northern Chinese population. Gene 710, 24–29. doi: 10.1016/j.gene.2019.05.011

mannose receptor recognition to regulation of immune responses. J. Immunol. 185, 929–942. doi: 10.4049/jimmunol.1000866

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

Copyright © 2020 Naqvi and Endsley. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) 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.

# A TLR2-Activating Fraction From *Mycobacterium abscessus* Rough Variant Demonstrates Vaccine and Diagnostic Potential

Vincent Le Moigne1 \*, Anne-Laure Roux 1 , Aude Jobart-Malfait 1 , Landry Blanc2 , Karima Chaoui 2 , Odile Burlet-Schiltz 2 , Jean-Louis Gaillard1 , Stéphane Canaan3 , Jérôme Nigou2 and Jean-Louis Herrmann1,4

1 Université Paris-Saclay, UVSQ, Inserm, Infection et inflammation, Montigny-le-Bretonneux, France, 2 Institut de Pharmacologie et de Biologie Structurale, Université de Toulouse, CNRS, Université Paul Sabatier, Toulouse, France, 3 Université Aix-Marseille, CNRS, LISM, IMM FR3479, Marseille, France, 4 APHP, GHU Paris-Saclay, Hôpital Raymond Poincaré, Service de Microbiologie, Garches, France

#### *Edited by:*

David Neil McMurray, Texas A&M Health Science Center, United States

#### *Reviewed by:*

Ashu Sharma, University at Buffalo, United States Ramon A. Juste, NEIKER Basque Institute for Agricultural Research and Development, Spain

> *\*Correspondence:* Vincent Le Moigne<EMAIL_ADDRESS>
#### *Specialty section:*

This article was submitted to Microbes and Innate Immunity, a section of the journal Frontiers in Cellular and Infection Microbiology

> *Received:* 28 January 2020 *Accepted:* 14 July 2020 *Published:* 27 August 2020

#### *Citation:*

Le Moigne V, Roux A-L, Jobart-Malfait A, Blanc L, Chaoui K, Burlet-Schiltz O, Gaillard J-L, Canaan S, Nigou J and Herrmann J-L (2020) A TLR2-Activating Fraction From Mycobacterium abscessus Rough Variant Demonstrates Vaccine and Diagnostic Potential. Front. Cell. Infect. Microbiol. 10:432. doi: 10.3389/fcimb.2020.00432 Mycobacterium abscessus is a prevalent pathogenic mycobacterium in cystic fibrosis (CF) patients and one of the most highly drug resistant mycobacterial species to antimicrobial agents. It possesses the property to transition from a smooth (S) to a rough (R) morphotype, thereby influencing the host innate immune response. This transition from the S to the R morphotype takes place in patients with an exacerbation of the disease and a persistence of M. abscessus. We have previously shown that the exacerbation of the Toll-like receptor 2 (TLR2)-mediated inflammatory response, following this S to R transition, is essentially due to overproduction of bacilli cell envelope surface compounds, which we were able to extract by mechanical treatment and isolation by solvent partition in a fraction called interphase. Here, we set up a purification procedure guided by bioactivity to isolate a fraction from the R variant of M. abscessus cells which exhibits a high TLR2 stimulating activity, referred to as TLR2-enriched fraction (TLR2eF). As expected, TLR2eF was found to contain several lipoproteins and proteins known to be stimuli for TLR2. Vaccination with TLR2eF showed no protection toward an M. abscessus aerosol challenge, but provided mild protection in 1F508 mice and their FVB littermates when intravenously challenged by M. abscessus. Interestingly however, antibodies against TLR2eF compounds were detected during disease in CF patients. In conclusion, we show the potential for compounds in TLR2eF as vaccine and diagnostic candidates, in order to enhance diagnosis, prevent and/or treat M. abscessus-related infections.

Keywords: *Mycobacterium abscessus*, cystic fibrosis, lipoprotein TLR2, vaccine adjuvant, diagnosis

# INTRODUCTION

Mycobacterium abscessus, a rapid growing mycobacterium (RGM), is emerging as a difficult-to-treat pathogen in cystic fibrosis (CF) patients. Mycobacterium abscessus is responsible for muco-cutaneous (Viana-Niero et al., 2008) and pulmonary infections (Griffith et al., 1993; Olivier et al., 2003; Roux et al., 2009). Severe and often fatal infections have been described

and are able to persist for decades in selected infected hosts (Cullen et al., 2000; Jönsson et al., 2007; Catherinot et al., 2009; Qvist et al., 2015). Above all, it is also the most resistant bacterium to antibiotics, with well documented therapeutic failures (Sanguinetti et al., 2001).

Mycobacterium abscessus is able to transition from a smooth (S) to a rough (R) morphotype. This S to R transition is linked to genetic insertions/deletions (Pawlik et al., 2013), which results in the arrest of synthesis and/or transport of glycopeptidolipids (GPL) at the cell surface (Ripoll et al., 2007). This event, occurring throughout the course of the disease, modifies completely the intensity of the inflammatory response triggered by M. abscessus S or R in vivo (Catherinot et al., 2007, 2009). The loss of GPL in the R variant is compensated by the increased synthesis and exposure at the cell surface of numerous lipoproteins, such as LpqH, LprA, and LprG (Roux et al., 2011). After isolating a fraction enriched in lipoproteins from the cell surface of the M. abscessus R variant, we found this fraction was responsible for TLR2 activation pathway provoking an important inflammatory response (Roux et al., 2011). This cell envelope remodeling was responsible for the TLR2-mediated hyper-pro-inflammatory phenotype of virulent R variants (Roux et al., 2011).

Major TLR2-activators are found among mycobacterial compounds, including lipoproteins (Brightbill et al., 1999; Thoma-Uszynski et al., 2000, 2001), lipomannan (LM) and phosphatidylinositol-mannosides (PIMs) (Gilleron et al., 2003; Quesniaux et al., 2004), in addition to several other proteins, including some heat shock proteins (HSP) (Jo et al., 2007).

The immune response triggered by the various mycobacterial glycolipids or lipoproteins in antigen-presenting cells is balanced, depending on the pattern recognition receptors (TLRs, DC-SIGN, Mannose Receptor, . . . ) engaged, between a protective inflammatory response characterized by the synthesis of IL-12 and Interferon gamma (IFNG), and a response with antiinflammatory IL-10 release leading to an impaired T cell response (Nigou et al., 2001; Tailleux et al., 2002; Geijtenbeek et al., 2003; Kaufmann and Schaible, 2003).

The use of TLR activation strategies induced by lipoproteins was previously proposed in the context of vaccine development, with the goal of amplifying antibacterial defense mechanisms and immune responses (Brightbill et al., 1999). TLR agonists have potent immunomodulatory activities and can promote adaptive immune responses to co-administered antigens. Consequently, they have been exploited as adjuvants in vaccines for other infectious diseases and cancers as anti-tumor immunotherapy (Conroy et al., 2008; Higgins and Mills, 2010).

In the present study, we evaluated both the vaccine and diagnostic potential in M. abscessus infection of a TLR2 activating fraction purified from M. abscessus R variant.

# MATERIALS AND METHODS

#### Bacterial Strain Culture

Mycobacterium abscessus R variant was grown in 7H9 medium containing 1% glucose and cells were collected by centrifugation. Pellets were weighted and conserved at −30◦C until extraction. For bacterial experimental infections, batches were prepared after centrifugation by washing pellets with phosphate-buffered saline (PBS), centrifuged and finally suspended in PBS with 10% glycerol, and dissociated by repeated passage through a 29.5 gauge needle. Then, 1-ml aliquot bacterial batches were frozen and conserved at −80◦C until they were required and titrated.

#### Fraction Extraction and Purification by Reverse-Phase Chromatography

Mycobacterium abscessus pellets (40 g) were delipidated by several extractions with CHCl3/CH3OH. The first one with 180 mL CHCl3/CH3OH (1:2, v/v) and incubated 150 min at 50◦C in a glass jar with agitation as previously described (Nigou et al., 1997). The suspension was then centrifuged 15 min at 800 g at room temperature and the supernatant (lipids) was removed. The pellet was suspended in 200 mL of CHCl3/CH3OH (1:1, v/v) and incubated for 90 min as previously. After a second centrifugation (15 min at 800 g, room temperature), the supernatant was removed. The pellet was resuspended in 200 mL CHCl3/CH3OH (1:1, v/v) and shacked overnight at room temperature. After centrifugation, the supernatant was added to the two previously supernatants. Delipidated cells were then disrupted by sonication. Proteins were further extracted by refluxing the broken cells in 160 mL of 50% ethanol at 65◦C for 90 min. The supernatant was recovered and, following the same procedure, a second extraction was then performed on the remaining pellet. Finally, both supernatants were mixed. The resulting ethanol/water extract was evaporated with a Rotavap up to 4 mL final volume and to allow to solubilize proteins, eventually stuck on the glass tube, the flask was rinsed with 4 mL of water and sonicated. A final volume of 8 mL was then recovered, frozen and lyophilized. DNAse and RNAse digestion was then performed onto the dried extract in 3 mL of 20 mM Tris-HCl, 1 mM MgCl2, pH 7.5 containing 130 U of DNAse and 130 U of RNase (8 h at 37◦C). Dialysis (MW cut-off 6–8 kDa) against water was then performed followed by α-amylase digestion to remove glycans in a phosphate buffer 50 mM (pH 7.5) with 2% (w:w) for the enzyme (8 h, room temperature). A second dialysis (MW cut-off 6–8 kDa) against water was then performed. A total of 20 mg protein was obtained as determined by Bradford assay.

The protein fraction was resuspended at a concentration of 1 mg/mL in water and loaded onto a PuriFlash C4 bonded silica column (15µm particles, porosity 200 Å; Interchim) preequilibrated with Limulus Amoebocyte Lysate (LAL) tested water containing 0.1% Trifluoroacetic acid (TFA). A first wash of the column with 10 mL of water/0.1% TFA was then performed. The charged column was then placed onto the chromatograph in which all of the pipes have been filled previously with eluents, firstly 99% acetonitrile/water LAL 1%/0.1% TFA (buffer B), then 99.9% LAL water/0.1% TFA (buffer A). The gradient used to elute the different products of interest, with a flow rate of 2 mL/min, was: from 100% buffer A to 25% buffer A/75% buffer B in 15 min; 25% buffer A/75% buffer B to 100% buffer B in 10 min; 100% buffer B for 10 min; 100% buffer B to 100% buffer A in 5 min; 100% buffer A for 5 min. About 1 mL of the fractions from the final volume were collected in 90 glass tubes for 45 min to complete elution. Glass tubes were previously treated at 160◦C for at least 2 h to remove any traces of LPS. Collected fractions of 1 mL were dried in a speedvac, resuspended in 110 µL of water, and protein concentration was determined.

#### HEK-TLR2 Experiments

As described previously (Roux et al., 2011), HEK-BlueTM-TLR2 cells (InvivoGen, France), derivatives of HEK293 cells that stably express the human TLR2 and CD14 genes along with a NF-κBinducible reporter system (secreted alkaline phosphatase), were maintained in Dulbecco's modified Eagle's medium (DMEM, Gibco) containing 10% Fetal Bovine Serum (FBS, Gibco), 4.5 g/L glucose, 2 mM L-glutamine, 100 U/mL penicillin, 100µg/mL streptomycin (Sigma). Protein fraction (0.1 µL) and HEK-TLR2 cells (5 × 104 cells per well) were added to 96-well plates. A cells stimulation control was realized with the synthetic triacylated lipopeptide Pam3CSK4 in a range from 0.01 to 100 ng/mL. Reporter cells were stimulated for 18 h and alkaline phosphatase activity was measured by mixing 20 µL of the culture supernatant and 180 µL of Quanti-BlueTM (InvivoGen) and reading O.D. at 630 nm.

### Human Monocytes Derived Dendritic Cells (DCs) and Cytokines Quantification

Peripheral blood mononuclear cells were isolated from freshly collected blood samples obtained from healthy voluntary blood donors (Ambroise Paré Hospital, France) by density gradient centrifugation using a lymphocyte separation medium (GE Healthcare Bio-Sciences, Sweden) as previously described (Dulphy et al., 2007; Talpin et al., 2014). Monocytes were purified by positive selection using anti-CD14-coated magnetic micro beads (Miltenyi Biotec, Bergisch Gladbach, Germany). Sorted monocytes were morphogically homogeneous with 90% of CD14+ cells, as determined by flow cytometry. Monocytes were differentiated into DCs for 7 days in RPMI medium supplemented with 10% FCS 100 U/mL penicillin, 100µg/mL streptomycin, 500 UI/mL GM-CSF, and 500 UI/mL IL-4 (Miltenyi Biotec, Germany R&D Systems, Abingdon, UK). At day 2, fresh medium supplemented with GM-CSF and IL-4 was added to culture and supplemented with IL-4 only at day 6. For stimulation assays, cells were harvested and cultured for an additional 2 days in fresh culture medium supplemented with either Escherichia coli LPS (250 ng/mL, Sigma, Saint-Louis, MI, USA) or with various concentrations of the TLR2eF (0.1– 10µg/mL). Supernatants from immature DCs stimulated with or without bacterial products were harvested at 48 h as previously described (Dulphy et al., 2007). TNF-α was quantified by ELISA with kits provided by R&D Systems (Abingdon, UK).

#### Tandem Mass Spectrometry Analysis

Protein samples were partially air-dried in a speed-vac device, reconstituted in 1× final Laemmli buffer, and loaded on a onedimensional SDS-PAGE gel. The electrophoretic migration was stopped as soon as the proteins entered the separating gel, in order to isolate all proteins in a single gel band. This band was washed, subjected to in-gel tryptic digestion and analyzed by nanoLC-MS/MS using an UltiMate 3000 RSLCnano system (Dionex, Amsterdam, The Netherlands) as previously described (Martinez-Pinna et al., 2014) except that peptides were eluted using a 5–50% gradient of solvent B (80% acetonitrile, 0.2% formic acid) during 5 h at 300 nL/min flow rate. The nanoLC system was coupled to an Orbitrap FusionTM TribridTM Mass Spectrometer (Thermo Scientific, Bremen, Germany) operated in a data-dependent acquisition mode with the XCalibur software. Survey scan MS were acquired in the Orbitrap on the 300– 2,000 m/z range with the resolution set to a value of 120,000. During data dependent acquisition, Orbitrap survey spectra were scheduled for execution at least every 3 s, with the embedded control system determining the number of MS/MS acquisitions executed during this period. Dynamic exclusion was employed within 60 s to prevent repetitive selection of the same peptide. Quadruplicate technical LC-MS measurements were performed.

#### Bioinformatic Processing of Mass Spectrometry Data

Raw mass spectrometry files were processed with the MaxQuant software (version 1.5.0) for database search with the Andromeda search engine and for quantitative analysis. Data were searched for against M. abscessus entries of the Uniprot-Swissprot-TrEMBL protein database (October 2019 version). Carbamidomethylation of cysteines was set as a fixed modification, whereas oxidation of methionine, protein Nterminal acetylation was set as variable modifications. Specificity of trypsin digestion was set for cleavage after K or R, and two missed trypsin cleavage sites were allowed. The precursor mass tolerance was set to 20 ppm for the first search and 5 ppm for the main Andromeda database search. The mass tolerances MS/MS mode was set to 0.6 Da. Minimum peptide length was set to seven amino acids, and the minimum number of peptides was set to one. Andromeda results were validated by the target-decoy approach using a reverse database at a both a peptide and protein FDR of 1%. The iBAQ metric, which corresponds to the sum of all the peptide intensities divided by the number of observable peptides of a protein (Schwanhäusser et al., 2011), was used to estimate absolute protein abundance. A threshold of iBAQ >6 × 108 was applied as it corresponds to an abrupt stall of the slope of the curve of iBAQ values.

# Vaccination and Infectious Challenge

Animal experiments were performed as previously described (Le Moigne et al., 2015), according to institutional and national ethical guidelines and approved by the Comité d'éthique en experimentation animale No. 047 with agreement A783223 under the reference APAFIS#11465. Briefly, 1F508 FVB mice and their wild type FVB littermates (van Doorninck et al., 1995) (INRA, Jouy en Josas, France) were housed in a bio-confinement level 2 facility and CF mice supplemented with movicol (Norgine, The Netherlands). Mice were immunized by subcutaneous (SC) injection with 20 µg of the TLR2eF diluted in PBS in a final volume of 200 µL following a prime/boost scheme (days 0, 21, and 42) as described (Le Moigne et al., 2015). Control mice received PBS only. Two weeks after the third immunization, mice were challenged by aerosol (4 × 108 bacterial cells/mL of CIP S) or intravenously (IV) (106 bacteria/mice) as previously described (Catherinot et al., 2007; Bernut et al., 2014; Bakala N'Goma et al., 2015). At various time-points post infection (Day 1, 12, and 21 for the aerosol challenge and Day 1 and 26 for the IV challenge), mice were sacrificed, and CFU counts were performed on lungs, spleen, and liver as described previously (Le Moigne et al., 2015). For each time-point, a total of 5–7 mice were infected. A control group of mice were sacrificed on the first day after infection (D1) allowing checking the inoculum. Blood samples were collected from the retro-orbital sinus for measurement of antibody levels by an ELISA approach. Samples were warmed at 37◦C for 1 h and then centrifuged (10,000 g, 10 min). Collected sera were stored at −80◦C until use.

#### ELISA Assay

Plates were coated with 1µg/mL of TLR2eF in 100 µL of carbonate-bicarbonate buffer (0.1 M, pH 9.6) overnight at 4◦C. Plates were then washed with PBS-Tween 20 (PBS-T) (0.05% v/v) and blocked with PBS-T containing 0.5% bovine serum albumin (PBS-T-BSA) 1 h at 37◦C. Sera previously obtained (Roux et al., 2009) were added at dilution of 1:400 in PBS-T-BSA, and were incubated 90 min at 37◦C. After four washes, goat anti-human total IgG alkaline phosphatase-conjugated (Southern biotechnology, Birmingham, USA) was added and plates were incubated again 90 min at 37◦C. After four washes, 100 µL of 1 mg/mL of p-nitrophenylphosphate (Sigma, Saint Quentin Fallavier, France) in diethanolamine buffer (pH 9.8) was added, and incubated at room temperature in the dark for 2 h. Plates were read at 405 nm with a Biorad PR 3100 TSC instrument (Biorad France, Marnes-la-Coquette, France).

### Western-Blot

Fifteen µg of TLR2eF per well with Laemmli buffer was electrophoresed on a 15% acrylamide SDS-PAGE. The gel was then transferred on a nitrocellulose membrane. After blocking with TBS-5% skimmed milk, the membrane was cut into bands corresponding to each well and each band was incubated for 2 h with a serum from a CF patient or a control serum diluted 1/500th in TBS-Tween 20 (0.1% v/v)−1% milk. After three washes in TBS-Tween, strips were incubated for two additional hours with goat anti-human IgG-HRP conjugated (CliniSciences, France). After three new washes, peroxidase activity was revealed with HRP substrate (WesternBright ECL, Advansta, CA, USA).

#### Statistical Analysis

For mice experiments, where two groups were compared, Student's t-test was used. A p-value < 0.05 was considered to be significant (ns = non-significant, ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001). In ELISA experiments, for comparison of the mean values of multiple groups, data that appeared to be statistically significant were compared by analysis of variance and non-parametric analysis using GraphPad Prism 6.0 software (GraphPad Software, La Jolla, CA, USA). A p-value < 0.05 was considered to be significant. (ns = non-significant, ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001). The technical values of the test in terms of sensitivity and specificity were calculated on the basis of ROC curves.

#### RESULTS

## Purification of a Pro-inflammatory TLR2-Activating Fraction

Mycobacterium abscessus S and R variants differ in the magnitude of the TLR2-mediated inflammatory response they trigger. We used M. abscessus R cells to extract cell wall components and to purify, using a cell-based bioassay, the most stimulating TLR2 fractions by reverse-phase chromatography. Elution fractions (EF) were tested for their ability to induce TLR2 signaling in a reporter cell line and their protein concentration was measured in parallel (**Figure 1A**). EFs 48–59 show the highest TLR2 stimulating activity, while containing a low amount of proteins, as shown in **Figure 1A**. These EFs were then pooled to allow subsequent experiments to be carried out. This pooled fraction (called TLR2eF for TLR2-enriched fraction) was separated by SDS-PAGE, showing an enrichment in 4 major proteins (indicated by a star) (**Figure 1B**). We next confirmed that TLR2eF was capable of inducing in a dose-dependent fashion the production of TNF-α by human dendritic cells (**Figure 1C**).

# Protein Composition of TLR2eF

We previously showed that the surface fraction overexpressed in M. abscessus R variant was enriched in lipoproteins (Roux et al., 2011). To identify the main proteins found in TLR2eF, we performed a proteomic analysis by mass spectrometry of (i) TLR2eF as a whole (**Table 1** and **Supplementary Table 1**), and (ii) the proteins under the four predominant bands illustrated in **Figure 1B**, after their extraction from the SDS-PAGE. Proteomic analysis of the whole TLR2eF identified many proteins, among which the first thirty most abundant, as determined by calculation of the iBAQ metric, are listed in **Table 1**. Three lipoproteins, namely LpqN (MAB\_4924) and LprG (MAB\_2806) and the conserved 19 kDa lipoantigen family protein (MAB\_0885c), were detected. Importantly, it is worth noting that tryptic digestion of lipoproteins is always suboptimal; therefore, the relative abundance of the lipoprotein in such analysis is often underestimated relatively to non-acylated proteins. Several potential lipoproteins or putative lipoprotein precursors of M. abscessus were also identified: LppK (MAB\_2160c), LprB (MAB\_1416), LpqE (MAB\_0567c), and three potential lipoproteins LpqH precursors (MAB\_0885c, MAB\_2379, and MAB\_3261c) (**Table 1**). Another putative lipoprotein belonging to the imelysin family protein (MAB\_1162c) and the putative lipoprotein MAB\_3983c, have also be identified. Mycobacterium abscessus homologs of other Mycobacterium tuberculosis proteins described to induce signaling via TLR2 were also found abundant in TLR2eF, such as Hsp65, Hsp70, and GroEL (**Table 1**) (Floto et al., 2006; Jo et al., 2007; Heo et al., 2011; Basu et al., 2012). Although described as TLR2 ligands (Basu et al., 2007; Palucci et al., 2016), no PE\_PGRS proteins were detected in TLR2eF, albeit one WXG100 type VII secretion target family protein (MAB\_3754c; EsxU) was observed (**Table 1**). Finally, protein representatives of the Ag85 complex (85A and 85C), in addition to a porin (MSPA; MAB\_1080) were also detected, although nothing is presently known regarding their potential recognition by TLR2. Analysis of

measured in the culture supernatant after 48 h of incubation.

the four individual bands excised from the gel identified many of the proteins listed in **Table 1** and did not allow to assign a specific protein to each band (not shown), because of the still complex mixture of proteins composing TLR2eF.

### TLR2eF Provides a Partial Protection Against an *M. abscessus* IV Challenge in Mice

Mice were vaccinated three times (Day 1, 21, and 42) subcutaneously (SC) at doses of 20 µg of TLR2eF, and then challenged 2 weeks after the third immunization, as previously described (Le Moigne et al., 2015). 1F508-CFTR mutated mice (1F508-FVB) and their wild-type littermates (FVB mice) were challenged either by aerosol (**Figure 2A**) or by the intravenous (IV) routes (**Figure 2B**) by M. abscessus (Le Moigne et al., 2015). Mycobacterium abscessus CFU counts were measured per organ (liver, spleen, and lung) at different time points (**Figure 2**).

After an aerosol challenge, we could not see any significant difference in the numbers of viable bacteria in lungs in all evaluated mice (**Figure 2A**). As previously described (Bernut et al., 2014; Le Moigne et al., 2015), CFU in mouse spleen and liver after an aerosol infection were below the sensitivity limit to be counted.

In contrast, M. abscessus challenge by an intravenous route allowed recovery of sufficient CFUs in all organs. At day 1

#### TABLE 1 | Proteins detected in TLR2eF by mass spectrometry.

\*Lipoproteins.

post-infection the CFU count was comparable in all organs. However, at day 26 post-infection, TLR2eF-vaccinated FVB and 1F508-FVB mice had significantly fewer mycobacteria in the liver (P ≤ 0.05) and in the lungs (P ≤ 0.05) as compared to non-vaccinated control FVB and 1F508-FVB mice (**Figure 2B**). These results suggest that TLR2eF administration before infection, can induce partial protection against M. abscessus IV challenge in mice.

FIGURE 2 | Bacterial load of M. abscessus CIP S after aerosol (A) or IV (B) infections in liver, spleen, and lungs of TLR2eF vaccinated or control-PBS vaccinated wild-type FVB and 1F508-FVB mice. Lungs, spleen, and liver of mice, infected with an aerosolized solution containing 4 × 108 bacteria/mL of CIP S or intravenously infected with 106 bacteria/mice, were collected and homogenized by dislocation. Homogenates were serially diluted and plated on VCAT medium plates for CFU count. Results are expressed as the log units of CFU for TLR2eF-vaccinated mice [yellow (A) and red (B) bars] or PBS vaccinated mice [control group, light blue (A), and blue (B) bars] at days 1, 12, and 21 post-infection for aerosol infection and days 1 and 26 for IV infection. Statistical values for FVB and 1F508-FVB were; P = 0.019 and P = 0.033, respectively in their liver; P = 0.374 and P = 0.298, respectively in the spleens and P = 0.023 and P = 0.045, respectively in the lungs. For each time-point, a total of 5–7 mice were infected (ns = non-significant, \*P < 0.05).

# TLR2eF Is Recognized by Mice and Human Sera

TLR2eF was further used as an antigen source in an ELISA assay aimed at diagnosis of M. abscessus infection. Firstly, the intensity of the antibody response generated by TLR2eFimmunized mice was compared to the response generated in the M. abscessus phospholipase C (PLC)-vaccinated mice (Le Moigne et al., 2015). As a negative control of the antibody response, we used mouse sera sampled at day 1 post-immunization. As shown in **Figure 3A**, we were able to detect an antibody response against TLR2eF in vaccinated mice. However, the intensity of the antibody response was inferior to the intensity of the antibody response obtained in PLC-vaccinated mice (**Figure 3A**).

We then evaluated the antibody response in human sera from CF patients, either infected with non-tuberculous mycobacterial (NTM) or not (**Figure 3B**). In order to develop the test, we have chosen from the collection of sera from a prevalence survey carried out in a population of CF patients in France (Roux et al., 2009), 10 CF patients culture positive for M. abscessus, five CF patients culture positive for Mycobacterium avium and five CF patients culture positive for Mycobacterium intracellulare. As a control population, we selected 20 CF patients from the same study who were NTM negative, and Pseudomonas aeruginosa positive or negative in culture. The serological response for patients infected with NTM was higher than in those NTM uninfected or infected with another bacterium such as P. aeruginosa (P < 0.001 and P < 0.0001, respectively) (**Figure 3B**). Nevertheless, when the NTM group is separated according to the mycobacterial species, we observe a true separation between positive and negative by this serological approach, from the population of CF-positive patients in culture to M. abscessus or M. intracellulare as compared to M. avium (**Figure 3C**); with six patients notably showing a response similar to that observed in control patients (4 out of 5 M. avium, 1 out of 5 M. intracellulare,

CF patients not infected by non-tuberculous mycobacteria, and infected or not by Pseudomonas aeruginosa, respectively. Results are mean ± SEM and were analyzed by two-way ANOVA. \*\*\*P < 0.001; \*\*\*\*P < 0.0001. (C) Specific anti-TLR2eF antibody response in sera from cystic fibrosis patients. Same results as in (B) but "NTM" group is splitted in three groups according NTM infection: CF patients infected by M. abscessus (red circles), infected by M. intracellulare (purple circles), and by M. avium (yellow circles). "Non-NTM Pa+" (green squares) and "Non-NTM Non-Pa" (blue triangles) are as in (B). Results are mean ± SEM and were analyzed by two-way ANOVA. ns, non-significant, \*P < 0.05; \*\*\*P < 0.001; \*\*\*\*P < 0.0001. (D) Anti-TLR2eF antibody response of sera from cystic fibrosis patients observed in Western Blot. Sera from seven of the best CF patients responders (P#1–P#7) against TLR2eF in ELISA were diluted at 1/500th and tested in western-blot against 15 µg of TLR2eF resolved on a 15% acrylamide gel and transferred to a nitrocellulose membrane. Serum from seven non-CF persons (C1–C7) were used as a control.

and 1 out of 10 M. abscessus). The technical values of the test in terms of sensitivity and specificity were calculated on the basis of ROC (not shown, AUC = 0.779) curves allowing the determination of a threshold value of 1.803. The test sensitivity was 70% (95% CI 45.7 to 88.1%) and the specificity 95% (95% CI 75.1 to 99.9%).

In order to determine by western blot which proteins in TRL2eF might be recognized by CF patient antibodies, we used seven positive CF sera, used in the ELISA assay, as examples. Although two proteins with apparent molecular weights between 17 and 20 kDa, which might correspond to one of the major proteins observed in SDS-PAGE (**Figure 1B**), were commonly recognized in six tested sera (indicated by two stars in **Figure 3D**), each serum showed a different pattern.

### DISCUSSION

Mycobacterial species that are pathogenic for humans and animals have a specific ability to both positively and negatively regulate innate and adaptive immune responses in the host (Kaufmann, 2001; Abel et al., 2002). One deleterious consequence is the persistence of pathogens within the infected host. Pathogenic mycobacteria have turned this to their own advantage, striking a balance with their hosts, and making it difficult or even impossible to eradicate (Zahrt, 2003). The peculiar mycobacterial cell envelope plays a key role in such modulation. The outer layers, composed of the capsule and the mycomembrane, confer protection against antiseptics and antibiotics, and contain many (glyco)lipids that are inflammatory triggers, although some other (glyco)lipids might modulate the intensity of the response via anti-inflammatory or masking properties.

Mycobacterium abscessus possesses the advantageous property of an interchangeable morphology, as described for some other mycobacterial species in vitro (Fregnan and Smith, 1962). However, the M. abscessus transition from S to R morphology was mainly observed in vivo, during infection in CF patients (Catherinot et al., 2009), or in mouse models (Rottman et al., 2007). The consequences are often catastrophic for CF patients and result in exacerbations of disease with acute respiratory distress syndrome (Catherinot et al., 2009). The main pathological mechanisms are (i) a hyper-proinflammatory response, due a very high TLR2 activation and the synthesis of proinflammatory cytokines and (ii) a very rapid extracellular growth (Salvator et al., 2018) with the formation of cords (Bernut et al., 2014).

In order to combat infection with this multi-resistant mycobacterium, our goal here was to evaluate the vaccine and diagnostic potentials of a still crude cell envelope fraction, TRL2eF, showing a high TLR2-stimulating activity. Although the serological response observed was of interest, the protective response after immunization was disappointing.

Despite being prepared using different techniques, TRL2eF and the so-called interphase fraction reported in Roux et al. (2011) show a similar protein composition, most particularly regarding lipoproteins. Of course, the composition of such crude fractions may be slightly different from one batch to another, but our first goal was to prove the usefulness and the potency of the TLR2-stimulating compounds overexpressed in the M. abscessus R variant, as a whole fraction.

TLR2eF showed no protection toward an aerosol challenge, and only a partial protection in the liver and the lungs toward an IV M. abscessus challenge. In fact, immunization with the TLR2eF, might be insufficient to activate the adaptive immune response required to make a vaccine, as we also need potent antigens. Consequently, do we confer an antigen-dependent protection or simply boost innate immunity that results in an improvement in infection outcome via the IV route only?

Signals from TLR2 are required for phagosome maturation (Blander and Medzhitov, 2004) and reduction of M. tuberculosis viability inside macrophages (Thoma-Uszynski et al., 2001). However, the intense inflammatory response observed in CF patients infected with M. abscessus during the emergence of the R variant (Catherinot et al., 2009), rather results in a lack of infection control. The TLR2-induced response might be suboptimal due to the presence of TLR2 lipid antagonists in TLR2eF. For instance, complex M. tuberculosis glycolipids have been described as TLR2 antagonists (Blanc et al., 2017), but such lipids have been removed during the purification procedure of TLR2eF (see experimental section). We cannot exclude the presence of TLR2 protein antagonists in TLR2eF, as described for ESAT-6 (EsxA), which inhibits TLR2-mediated NF-kB and IRFs activation (Pathak et al., 2007). In fact, we found a protein of this ESX family in the main proteins of our TLR2eF: EsxU (MAB\_3754c). A Tn library screening, recently described for M. abscessus (Dubois et al., 2018; Laencina et al., 2018) and M. tuberculosis (Blanc et al., 2017) might allow a more precise characterization of such antagonists.

Finally, we argue that the immune response required to control bacterial infection might be different with regard to the route of M. abscessus administration. A B-cell response might preferentially act to inhibit bacterial respiratory infection in mice, through the secretion of immunoglobulin-A (IgA) in the lungs. However, lipoprotein inhibition of MHC-II presentation might be responsible for the induction of a suboptimal B cell response (Harding and Boom, 2010). As we have seen a weaker antibody response after TLR2eF immunization in mice, as compared to PLC vaccinated mice, the fact that we did not observe protection after aerosol challenge could be due to the partial inhibition of this humoral response, an important factor to circumvent pathogen invasion in the context of lung infection. Comparatively, a predominant antigen-specific CD8+ T cell (CTL) response is observed in the systemic compartment, when boosted by TLR2-ligands (Deres et al., 1989; Jackson et al., 2004; Borsutzky et al., 2006). This might explain the more rapid elimination of M. abscessus seen repeatedly in the TLR2eFvaccinated IV infected mice.

Despite a disappointing protective response, it was interesting to evaluate whether individual components in TLR2eF were recognized by the immune system in CF patients infected with M. abscessus. This latter point was highlighted by a recent clinical study showing the importance of detecting M. abscessus infection in CF patients (Qvist et al., 2015). Therefore, clinical values obtained in our study are very promising for the detection of patients who have been in contact with NTM and have developed an antibody response. What is fundamentally interesting is that the lowest optical density values, similar to those of control patients, are obtained for patients infected with M. avium. Four of the six patients with the lowest values are infected with M. avium. By improving the technical values of our serological test, we will therefore be able to differentiate between CF patients infected with M. avium and those infected with M. abscessus. On the contrary, meeting the initial objective of having a reagent allowing to discriminate NTMs, the goal is reached. Additional studies should be undertaken to know if there is common antigens or TLR2-ligands between M. abscessus and M. intracellulare and inversely to explain the absence of response with M. avium. From a diagnostic point of view, it may make it possible to detect this patient group, and to better evaluate them clinically and microbiologically in order to adapt treatment to prevent progressive infection. In addition to detecting CF patients infected by M. abscessus, the presence of high antibody responses, as seen in several patients against TLR2eF, might allow the differentiation of CF patients infected with the R morphotype, responsible for the most severe form of the disease, and with an imperative for early treatment.

Based on this study, we will pursue our efforts to evaluate the use of TLR2eF as a serological tool, more particularly to determine its potential for identifying NTM infections in CF patients in the context of microbiological diagnosis during epidemiological studies.

#### DATA AVAILABILITY STATEMENT

All datasets generated for this study are included in the article/**Supplementary Material**.

#### ETHICS STATEMENT

The studies involving human participants were reviewed and approved by the OMA (Observatoire des mycobactéries

#### REFERENCES

atypiques) group. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by Comité d'éthique en experimentation animale N◦ 047 with agreement A783223.

# AUTHOR CONTRIBUTIONS

VL, J-LG, JN, and J-LH designed the project and experiments. VL, A-LR, AJ-M, LB, KC, and OB-S performed the experiments. VL, JN, SC, and J-LH wrote and corrected the manuscript. All authors contributed to the article and approved the submitted version.

#### FUNDING

This work was supported by grants from the Association Vaincre la Mucoviscidose (RF20110600446/1/3/130) and the Région Île-de-France (post-doctoral fellowship for VL).

# ACKNOWLEDGMENTS

We gratefully acknowledge Dr. Jacques Prandi and Ms. Sophie Zuberogoitia (IPBS) for helpful assistance with reverse phase chromatography. We also acknowledge Dr. B. Marshall (Southampton University, UK) for helpful comments and critical reading of the manuscript. This work has benefited from the facilities and expertise of the proteomic platform of IMM FR3479 CNRS.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcimb. 2020.00432/full#supplementary-material

production of TLR2 antagonist glycolipids. Proc. Natl. Acad. Sci. U.S.A. 114, 11205–11210. doi: 10.1073/pnas.1707840114

massiliense and Mycobacterium bolletii in isolates collected from outbreaks of infections after laparoscopic surgeries and cosmetic procedures. J. Clin. Microbiol. 46, 850–855. doi: 10.1128/JCM.02052-07

Zahrt, T. C. (2003). Molecular mechanisms regulating persistent Mycobacterium tuberculosis infection. Microbes Infect. 5, 159–167. doi: 10.1016/S1286-4579(02)00083-7

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

Copyright © 2020 Le Moigne, Roux, Jobart-Malfait, Blanc, Chaoui, Burlet-Schiltz, Gaillard, Canaan, Nigou and Herrmann. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) 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.

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