1. Immunology and Inflammation
  2. Microbiology and Infectious Disease
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Identification of scavenger receptor B1 as the airway microfold cell receptor for Mycobacterium tuberculosis

  1. Haaris S Khan
  2. Vidhya R Nair
  3. Cody R Ruhl
  4. Samuel Alvarez-Arguedas
  5. Jorge L Galvan Rendiz
  6. Luis H Franco
  7. Linzhang Huang
  8. Philip W Shaul
  9. Jiwoong Kim
  10. Yang Xie
  11. Ron B Mitchell
  12. Michael U Shiloh  Is a corresponding author
  1. Department of Internal Medicine, University of Texas Southwestern Medical Center, United States
  2. Center for Pulmonary and Vascular Biology, Department of Pediatrics, University of Texas Southwestern Medical Center, United States
  3. Quantitative Biomedical Research Center, Department of Population and Data Sciences, University of Texas Southwestern Medical Center, United States
  4. Harold C Simmons Cancer Center, University of Texas Southwestern Medical Center, United States
  5. Department of Bioinformatics, University of Texas Southwestern Medical Center, United States
  6. Department of Otolaryngology, University of Texas Southwestern Medical Center, United States
  7. Department of Microbiology, University of Texas Southwestern Medical Center, United States
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Cite this article as: eLife 2020;9:e52551 doi: 10.7554/eLife.52551

Abstract

Mycobacterium tuberculosis (Mtb) can enter the body through multiple routes, including via specialized transcytotic cells called microfold cells (M cell). However, the mechanistic basis for M cell entry remains undefined. Here, we show that M cell transcytosis depends on the Mtb Type VII secretion machine and its major virulence factor EsxA. We identify scavenger receptor B1 (SR-B1) as an EsxA receptor on airway M cells. SR-B1 is required for Mtb binding to and translocation across M cells in mouse and human tissue. Together, our data demonstrate a previously undescribed role for Mtb EsxA in mucosal invasion and identify SR-B1 as the airway M cell receptor for Mtb.

Introduction

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), latently infects roughly one-third of the world’s population and causes 1–2 million deaths per year. The current paradigm of acute infection is that after an actively infected person aerosolizes infectious Mtb-containing particles, a naive individual inhales the bacteria that then traverse the respiratory tree to ultimately be phagocytosed by alveolar macrophages (Churchyard et al., 2017; Cohen et al., 2018). While this model can account for pulmonary TB, it is insufficient to explain some extrapulmonary forms of TB initiated by oropharyngeal infection and lacking evidence of concurrent pulmonary disease. For example, a disease known as tuberculous cervical lymphadenopathy, or scrofula, represents 10% of all new cases of TB, and frequently manifests without lung involvement (Fontanilla et al., 2011). Because the oropharynx and upper airway lymphatics drain to the cervical lymph nodes, while the lower airway lymphatics drain to the mediastinal lymph nodes, infection of the cervical lymph nodes by Mtb may not involve the lower airways. Indeed, in the infamous ‘Lübeck Disaster’ where hundreds of infants and children were accidentally orally administered Mtb instead of the attenuated BCG vaccine, the majority developed lymphatic and oropharyngeal TB rather than pulmonary TB (Fox et al., 2016), highlighting how inoculation via the oropharyngeal route can cause extrapulmonary disease.

One potential explanation for the development of lymphatic TB centers upon the mucosa-associated lymphoid tissue (MALT) (Brandtzaeg et al., 2008). Specialized epithelial cells known as microfold cells (M cells) overlie the MALT and are able to translocate luminal material to basolateral antigen-presenting cells located immediately beneath the M cell (Kimura, 2018). In this way, M cells can initiate an immune response to pathogens or material found within the lumen (Nakamura et al., 2018).

Since their initial discovery overlying Peyer’s patches of the gastrointestinal tract, M cells have been identified at other mucosal sites. Within the respiratory tract, M cells have been found in the upper and lower airways of both mice and humans (Fujimura, 2000; Kim et al., 2011; Kimura et al., 2019). M cells express a number of pattern recognition receptors (PRRs) (Mabbott et al., 2013). The majority of these M cell receptors have been identified on gastrointestinal M cells, while receptor expression by airway microfold cells is less well understood. Some PRRs on gastrointestinal M cells function in bacterial recognition and translocation. For example, the cellular prion protein (PrP(C)), a receptor for Brucella abortus, is necessary for B. abortus translocation (Nakato et al., 2012). Similarly, glycoprotein 2 (GP2) expressed on the apical surface of gastrointestinal M cells recognizes FimH, a component of the type I pili found on both commensal and pathogenic bacteria (Hase et al., 2009). Loss of either the host receptor GP2 or the bacterial ligand FimH diminishes bacterial translocation through M cells, reducing the immune response to these antigens and bacteria within Peyer’s patches (Hase et al., 2009).

We previously demonstrated that Mtb uses airway M cells as a portal of entry to initiate infection (Nair et al., 2016). We hypothesized that Mtb may produce a bacterial effector to mediate this process, and that, similar to receptors for gram-negative bacteria in the gastrointestinal tract (Hase et al., 2009), airway M cells may also encode an Mtb receptor. Here, we show that Mtb requires the type VII secretion system for translocation in vitro and in vivo. The type VII secretion system effector EsxA (also known as ESAT-6) is sufficient to mediate this process in vitro through binding to scavenger receptor class B type I (SR-B1). SR-B1 is enriched on mouse and human M cells both in vitro and in vivo. Loss of SR-B1 reduces EsxA and Mtb binding to M cells, and prevents Mtb translocation through M cells in vitro. Using a newly developed explanted human adenoid model, we demonstrate robust expression of SR-B1 on primary human M cells. Finally, we show that Mtb infects primary human M cells on adenoids in a type VII secretion system dependent manner. Taken together, our findings indicate that the interaction of Mtb EsxA with M cell SR-B1 allows Mtb to traverse the airway mucosa to initiate infection.

Results

The Mtb type VII secretion system mediates Mtb binding to and translocation through M cells in vitro

Mtb encodes several protein secretion systems important for bacterial virulence (Feltcher et al., 2010). One of the type VII secretion systems (T7SS) of Mtb, contained within the region of difference 1 (RD1) locus of Mtb (Behr et al., 1999), secretes virulence factors including EsxA and EsxB (also known as CFP-10) (Stanley et al., 2003; Figure 1A). We hypothesized that the Mtb T7SS might facilitate M cell translocation because the T7SS machine interacts directly with eukaryotic membranes (Augenstreich et al., 2017), EsxA can be identified on the mycobacterial cell surface (Kinhikar et al., 2010), and EsxA may directly bind several cell surface receptors (Kinhikar et al., 2010; Sreejit et al., 2014). To test if the Mtb T7SS was required for bacterial binding to and translocation across M cells, we used a human airway M cell transwell model that we developed previously (Figure 1B; Nair et al., 2016). Such transwells combine 16-HBE cells (Cozens et al., 1994) in the apical compartment of a transwell and Raji B cells in the basolateral compartment in order to mimic the organization of MALT and to enhance M cell formation (Kernéis et al., 1997; Nair et al., 2016). HBE cells cultured alone form a homogenous, polarized monolayer (hereafter called ‘control’) while coculture with Raji B cells induces some HBE cells to differentiate into M cells (hereafter called ‘HBE/Raji B’) (Nair et al., 2016). We used the Mtb eccD1::Tn5370 strain (hereafter designated Mtb eccD1::Tn) (Cox et al., 1999; Stanley et al., 2003), which lacks the inner membrane pore required for assembly of and protein secretion by the T7SS (Figure 1A; Abdallah et al., 2007). To test if the Mtb T7SS was necessary for M cell binding, we incubated transwells with either wild-type Mtb expressing mCherry (WT Mtb) or Mtb eccD1::Tn expressing mCherry at 4°C to prevent bacterial entry or translocation and analyzed surface binding by confocal microscopy and quantification of colony-forming units (CFU) (Figure 1C–E). Consistent with our prior data (Nair et al., 2016), significantly more WT Mtb bound HBE/Raji B transwells (containing M cells) than control transwells by both microscopy (Figure 1C,D) and CFU (Figure 1E). However, binding was greatly reduced for the Mtb eccD1::Tn strain (Figure 1C–E). To confirm that the Mtb eccD1::Tn strain did not harbor additional mutations other than the expected transposon insertion into eccD1 to potentially explain this result, we performed whole genome sequencing on the Mtb Erdman WT lab strain from our lab, along with the Mtb eccD1::Tn strain. No significant insertions, deletions or mutations were observed in the Mtb eccD1::Tn strain relative to the wild-type strain, although several single-nucleotide polymorphisms were noted when we compared the strains to the reference strain (Miyoshi-Akiyama et al., 2012; Supplementary file 1).

Mtb T7SS is necessary to mediate binding and translocation across M cells.

(A) Model of Mtb T7SS. (B) Schematic of human airway M cell transwell model. (C,D) Control and HBE/RajiB transwells were incubated with Mtb strains at 4°C for 1 hr and binding was analyzed by confocal microscopy (C) with quantification of bacterial number (D). Scale bar, 20 µm. (E) Control and HBE/RajiB transwells were incubated with Mtb strains at 4°C for 1 hr and lysed to determine binding by quantifying bacterial CFU and comparing with the initial inoculum. (F) Control and HBE/RajiB transwells were incubated with Mtb strains at 37°C for 1 hr and bacterial translocation was determined by quantifying bacterial CFU from the basal compartment and comparing with the inoculum. (G) TEER measurements from transwells from (F). (H) Caco-2/Raji B transwells were infected as described in F. (I) TEER measurements from transwells from (H). Experiments shown are representative of at least three independent experiments. *p<0.05, ***p<0.0005 as determined by one-way ANOVA. Where not shown, comparisons were not significant.

To test if the Mtb T7SS is necessary to facilitate mycobacterial translocation across M cells, we infected the apical chamber of transwells with either WT Mtb or Mtb eccD1::Tn and measured translocation to the basal compartment. As we reported previously (Nair et al., 2016), WT Mtb translocated across HBE/Raji B transwells to a greater extent than control transwells, while the translocation of the Mtb eccD1::Tn strain was significantly reduced (Figure 1F). Importantly, the transepithelial electrical resistance (TEER), a measure of epithelial monolayer integrity (Srinivasan et al., 2015), was stable during the experiment (Figure 1G). To further verify that Mtb T7SS is required for bacterial translocation across M cells in vitro, we also utilized an established model of M cell differentiation where Caco-2 cells, a human colonic epithelial cell line, are cultured with Raji B transwells to induce M cell differentiation (Nair et al., 2016). Similar to HBE/Raji B transwells, we observed that Mtb translocated across Caco-2/Raji B transwells in a T7SS-dependent manner (Figure 1H,I). Taken together, these data show that the Mtb T7SS is necessary for both binding to and translocation across M cells in vitro.

EsxA is sufficient to mediate binding to and translocation across M cells in vitro

Two of the most abundant T7SS secreted proteins are EsxA and EsxB (Berthet et al., 1998; Brodin et al., 2005; Champion et al., 2014); therefore, we hypothesized that one of these proteins might mediate Mtb binding and translocation. We expressed EsxA and EsxB as 6-His tagged constructs and purified the proteins from E. coli (Figure 2—figure supplement 1). We then conjugated recombinant EsxA, EsxB, or glycine (as a control) to fluorescent beads, added the beads to the apical chamber of transwells, and quantified bead translocation to the basal compartment by flow cytometry. EsxA-beads but not control beads translocated across HBE/Raji B transwells (Figure 2A) without disrupting the epithelial monolayer (Figure 2B). EsxA-beads, but not EsxB-beads or control beads, also translocated across Caco-2/Raji B transwells (Figure 2C).

Figure 2 with 2 supplements see all
Mtb EsxA is sufficient to mediate binding and translocation across M cells.

(A) Control and HBE/RajiB transwells were incubated with fluorescent beads coated with EsxA, EsxB, or glycine. Translocation was determined by comparing the number of beads in the basal compartment with the inoculum. (B) TEER measurements from transwells from (A). (C) Caco-2/Raji B transwells were treated as described in A. (D) Control and HBE/RajiB transwells were incubated with recombinant EsxA or EsxB and stained with NKM 16-2-4 (red) and an anti-6-His antibody (green). Scale bar, 30 µm. (E–G) Quantification of nuclei (E), NKM 16-2-4+ cells (F), and EsxA+ (G) cells from the transwells described in (D). (H) Quantification of NKM 16-2-4 staining on EsxA+ cells from the HBE/Raji B transwells described in (D). Experiments shown are representative of at least three independent experiments. *p<0.05, **p<0.005, ***p<0.0005 as determined by one-way ANOVA. Where not shown, comparisons were not significant.

To test if the ability of EsxA to mediate translocation was due to direct EsxA binding to M cells, we incubated transwells with recombinant 6xHis-tagged EsxA or EsxB and performed immunofluorescence microscopy using antibodies against 6x-His and α1,2-fucose (NKM 16-2-4; a marker for M cells [Nair et al., 2016; Nochi et al., 2007; Figure 2D]). While both groups of transwells had equal number of nuclei per field (Figure 2E), HBE/Raji B transwells had more NKM 16-2-4-positive M cells compared to control transwells (Figure 2F). We detected robust EsxA binding to M cells on HBE/Raji B transwells, in contrast to EsxB, which did not demonstrate significant binding (Figure 2D,G–H). Similar results were observed using an antibody against Sialyl LewisA (SLA), a different M cell marker (Giannasca et al., 1999; Nair et al., 2016; Figure 2—figure supplement 2). Taken together, these data demonstrate that EsxA, but not EsxB, directly binds the M cell surface and is sufficient to mediate translocation across M cells when conjugated to inert beads.

Scavenger receptor class B type one binds EsxA and is expressed on M cells in vitro

Because EsxA bound directly to the surface of M cells, we hypothesized that EsxA may engage a cell surface receptor. To affinity purify cell surface binding proteins, we performed a modified co-immunoprecipitation experiment using either EsxA or transferrin crosslinked to the TriCEPS reagent, a molecule that allows for the covalent cross-linking of a ligand and its receptor (Tremblay and Hill, 2017). We used Caco-2 cells for this experiment as they have been used extensively as a model for M cells in vitro (Tyrer et al., 2006) and because Caco-2/Raji B transwells behaved similarly to HBE/Raji B transwells in Mtb and EsxA translocation (Figure 1E,G and Figure 2A,C). Using this approach, we identified the interaction between transferrin (TRFE) and the transferrin receptor (TFR1) (Figure 3A, blue peptides; Supplementary file 2), proving the validity of this system. When cells were treated with EsxA, peptides for two proteins, apolipoprotein E (ApoE) and scavenger receptor class B type I (SR-B1) were enriched (Figure 3A, red peptides; Supplementary file 2). Because ApoE is a soluble protein (Huang and Mahley, 2014) while SR-B1 is a known cell surface molecule, we focused on SR-B1. For further verification, we performed a co-immunoprecipitation/biotin transfer experiment without the TriCEPS reagent. After incubation with biotinylated EsxA, completion of the biotin transfer assay, and subsequent immunoprecipitation with streptavidin-coated beads, western blotting with an anti-SR-B1 antibody detected an approximately 82 kD band consistent with the known observed molecular weight of glycosylated SR-B1 (Acton et al., 1996; Figure 3B).

SR-B1 is the M cell EsxA receptor.

(A) Volcano plot displaying peptides enriched when Caco-2 cells were treated either with transferrin (blue dots on left) or EsxA (red dots on right). Results are of a single experiment with three biologic replicates per condition. (B) Western blot using an anti-SR-B1 antibody (top) or an anti-actin antibody (bottom) of proteins enriched after HBE cells were incubated with biotinylated EsxA or control. (C) Control and HBE/RajiB transwells were stained with NKM 16-2-4 and an anti-SR-B1 antibody and analyzed by confocal microscopy. Arrows denote examples of double positive cells. Scale bar, 40 µm. (D,E) Quantification of the number of nuclei (D) or SR-B1+ cells (E) from the transwells described in (C). (F) Quantification of NKM 16-2-4 staining on SR-B1+ cells from the HBE/Raji B transwells described in (C). Experiments shown are representative of at least three independent experiments. ***p<0.0005 as determined by one-way ANOVA. Where not shown, comparisons were not significant.

We next determined if SR-B1 expression is specific for M cells or ubiquitously expressed by epithelial cells. We quantified colocalization of SR-B1 and NKM 16-2-4 by immunofluorescence microscopy in control and HBE/Raji B transwells (Figure 3C–F). While there was no difference in the number of nuclei per field on the transwells (Figure 3C), SR-B1 expression was higher on HBE/Raji B transwells (Figure 3C,E) and the majority of the SR-B1 positive cells were NKM+ M cells (Figure 3C,F). Taken together, we identify SR-B1 as a candidate EsxA receptor expressed on M cells in vitro.

Genetic disruption of SR-B1 limits EsxA binding to M cells

We next investigated whether SR-B1 is required for EsxA binding to M cells by exposing cells to recombinant EsxA in the presence or absence of SR-B1. We first transduced HBE cells with non-targeting (NT) or SR-B1 shRNA and observed a robust knock-down of SR-B1 in HBE cells transduced with the SR-B1 shRNA as compared to the NT shRNA (Figure 4A). HBE/Raji B transwells constructed from these cells had a similar number of M cells comparing NT and SR-B1 shRNA transwells (Figure 4B,C) and SR-B1 shRNA transwells showed a reduction in SR-B1 expression by immunofluorescence microscopy (Figure 4B,D). When we incubated these transwells with recombinant EsxA, absence of SR-B1 reduced the number of EsxA positive cells on HBE/Raji B SR-B1 shRNA transwells (Figure 4B,E). Additionally in HBE/RajiB NT shRNA transwells, the majority of EsxA-positive cells were SR-B1 positive (Figure 4B,F), suggesting that EsxA preferentially bound SR-B1 expressing M cells. Taken together, we identify SR-B1 as necessary for EsxA binding to M cells.

Loss of SR-B1 reduces EsxA binding and Mtb translocation through M cells.

(A) Western blot of SR-B1 (top) or beta-actin (bottom) of shRNA expressing HBE cells. (B) HBE/RajiB transwells with shRNA expressing HBE cells were incubated with biotinylated EsxA and stained with NKM 16-2-4 (red), anti-SR-B1 (cyan), and Alexa Fluor 488 conjugated streptavidin (green). Arrows denote examples of triple positive cells. Scale bar, 40 µm. (C–E) Quantification of the number of NKM+ (C), SR-B1+ cells (D) and EsxA+ cells (E) on transwells described from (B). (F) Quantification of SR-B1 staining on EsxA+ cells from HBE NT shRNA/Raji B transwells described in (B). (G,H) HBE/RajiB transwells with shRNA expressing HBE cells were incubated with mCherry Mtb and Mtb binding was analyzed by confocal microscopy (G) with quantification of bacterial number (H). Scale bar, 10 µm. (I) HBE/RajiB transwells with shRNA expressing HBE cells were incubated with Mtb strains at 4°C and lysed to determine binding by quantifying bacterial CFU and comparing with the initial inoculum. (J) HBE/RajiB transwells with shRNA expressing HBE cells were incubated with Mtb strains at 37°C and bacterial translocation was determined by quantifying bacterial CFU from the basal compartment and comparing with the inoculum. (K) TEER of the transwells from (J). (L) HBE/RajiB transwells with shRNA expressing HBE cells were incubated with Pseudomonas aeruginosa at 37°C and bacterial translocation was determined by quantifying bacterial CFU from the basal compartment and comparing with the inoculum. Experiments shown are representative of at least three independent experiments. *p<0.05, **p<0.005, ***p<0.0005 as determined by one-way ANOVA. Where not shown, comparisons were not significant.

Genetic disruption of SR-B1 reduces both Mtb binding to and translocation across M cells

To determine the role of SR-B1 in Mtb binding to M cells, we incubated HBE/Raji B transwells expressing NT or SR-B1 shRNAs with mCherry Mtb at 4°C for 1 hr and analyzed binding by confocal microscopy and CFU (Figure 4G–I). Loss of SR-B1 greatly reduced the number of bacteria bound to the HBE/Raji B transwells as determined by confocal microscopy (Figure 4G,H) and by quantification of CFU (Figure 4I). To determine the role of SR-B1 in Mtb translocation by M cells, we infected HBE/Raji B transwells expressing NT or SR-B1 shRNAs with Mtb in the apical compartment and measured translocation to the basal compartment. As expected from the reduced bacterial binding (Figure 4G–I), loss of SR-B1 also greatly reduced Mtb translocation in the HBE/Raji B transwells at 37°C (Figure 4J) with no impact on the TEER (Figure 4K). The reduced ability of Mtb to translocate across HBE/Raji B transwells expressing SR-B1 shRNA was not due to any intrinsic defect in translocation caused by SR-B1 deficiency as another airway pathogen, Pseudomonas aeruginosa, was able to translocate equally across NT and SR-B1 shRNA HBE/Raji B transwells (Figure 4L). Of note, P. aeruginosa does not encode a T7SS or EsxA homologue, suggesting that its translocation across M cells depends on unique bacterial and host factors. We thus conclude that SR-B1 is essential for the binding and translocation of Mtb via M cells in a process requiring the effector EsxA.

The Mtb type VII secretion system is necessary for Mtb translocation in mice

M cells are found in the upper and lower airways of mice and humans (Fujimura, 2000; Mutoh et al., 2016; Nair et al., 2016; Teitelbaum et al., 1999). We therefore determined if SR-B1 was expressed preferentially by primary M cells as compared to other epithelial cells using immunofluorescence microscopy. Mouse nasal-associated lymphoid tissue (NALT), a region enriched for M cells (Mutoh et al., 2016; Nair et al., 2016; Park et al., 2003), demonstrated robust SR-B1 staining on the surface of NKM 16-2-4 positive cells (Figure 5A). Importantly, we did not observe SR-B1+/NKM 16-2-4- cells, demonstrating that SR-B1 is specific for M cells in the NALT epithelia in vivo.

Figure 5 with 3 supplements see all
The Mtb type VII secretion system is necessary for Mtb entry in mice and humans.

(A) Mouse NALT sections were stained with NKM 16-2-4 and anti-SR-B1 antibodies and analyzed by confocal microscopy. Scale bar, top, 15 µm, bottom, 5 µm. (B,C) Mice were intranasally infected with either WT Mtb, Mtb eccD1::Tn (B), or MtbΔesxA (C). CFU was determined in the NALT on day 0 (left) or in the cervical lymph nodes on day 7 (right). Symbols represent CFU from individual animals (n = 8–10 per strain). ***p<0.0005 compared to WT by Mann-Whitney U test. (D) Human adenoid sections were stained with NKM 16-2-4 and anti-SR-B1 antibodies and analyzed by confocal microscopy. Scale bar, top, 15 µm, bottom, 5 µm. (E) Human adenoids were disaggregated, stained with NKM 16-2-4 and anti-EpCAM antibodies, and analyzed by flow cytometry. (F) Human adenoids were treated as in (E), stained with anti-SR-B1 or control IgG antibodies and analyzed by flow cytometry. Symbols represent adenoids from individual donor (F–H). **p<0.005, Wilcoxon matched pairs signed rank test. (G) Human adenoids were infected with GFP+ Mtb, disaggregated, immunostained and analyzed by flow cytometry to determine the proportion of GFP+ Mtb containing NKM+/EpCAM+ and NKM-/EpCAM+ cells. *p<0.05, Wilcoxon matched pairs signed rank test. (H) Human adenoids were infected with GFP+ Mtb or GFP+ Mtb eccD1::Tn. The percentage of GFP+ Mtb containing NKM+/EpCAM+ double positive cells was determined by flow cytometry. The Wilcoxon matched pairs signed rank test was used for comparison.

We and others previously demonstrated that NALT and airway M cells are a portal of entry for Mtb in mice (Nair et al., 2016; Teitelbaum et al., 1999). To determine if the Mtb T7SS is necessary for bacterial translocation in vivo, we performed NALT infections (Nair et al., 2016) with Mtb eccD1::Tn or MtbΔesxA. As described above for Mtb eccD1::Tn, to control for possible mutations outside the known esxA deletion, we performed whole genome sequencing on the Mtb∆esxA strain and determined that its sequence is essentially identical to the parental Mtb WT strain (Supplementary file 1). Of note, in both Mtb mutant strains the T7SS machine fails to assemble (Abdallah et al., 2007) thereby preventing T7SS-dependent virulence factor secretion. We infected mice intranasally with WT Mtb, Mtb eccD1::Tn (Figure 5B), or MtbΔesxA (Figure 5C) and enumerated CFU from draining cervical lymph nodes 7 days post-infection (Nair et al., 2016). Both the Mtb eccD1::Tn and Mtb∆esxA strains had 1.0–1.5 log fewer bacteria compared to WT Mtb in the cervical lymph nodes. This degree of attenuation was not observed when we infected mice intranasally with a Cor-deficient strain of Mtb (Mtbcor::Tn7) that is also attenuated in vivo (Zacharia et al., 2013; Figure 5—figure supplement 1). Taken together, these data demonstrate that the lower CFU recovered from cervical lymph nodes of mice infected with T7SS mutant Mtb strains may be due to reduced translocation across M cells.

Human adenoid M cells express SR-B1

Because TB is a human disease, we tested if primary human M cells can serve as a portal of entry for Mtb. The human adenoid is a MALT structure that contains M cells interspersed among the overlying epithelial cells (Fujimura, 2000). We first demonstrated that human adenoids contain SR-B1+/NKM+ M cells by immunofluorescence microscopy (Figure 5D), similar to our observations from mouse NALT (Figure 5A). We next confirmed the presence of M cells in human adenoids by flow cytometry (Figure 5—figure supplement 2 for gating strategy) and observed that approximately 10% of adenoid cells were EpCAM+/NKM- epithelial cells, while about 1% of the cells were EpCAM+/NKM+ double positive M cells (Figure 5E). When we analyzed SR-B1 expression using flow cytometry, we observed that approximately 20% of primary human M cells (marked as EpCAM+/NKM+ cells) were SR-B1 positive as compared to less than 2% of the other epithelial cells (EpCAM+/NKM-) (Figure 5F), verifying our observation that SR-B1 is expressed predominately by M cells in vivo.

Human adenoid M cells are a portal of entry for Mtb

To determine if primary human M cells can be a route of entry for Mtb, we infected human adenoids with GFP+ Mtb and quantified the number of GFP+ Mtb EpCAM+/NKM+ M cells versus GFP+ Mtb EpCAM+/NKM- epithelial cells by flow cytometry (Figure 5G, Figure 5—figure supplement 3 for gating strategy). The number of GFP+ Mtb containing EpCAM+/NKM+ M cells ranged from 0.1–8% while we were unable to identify any GFP+ Mtb containing EpCAM+/NKM- epithelial cells (Figure 5G), suggesting that M cells were a preferred route of entry for Mtb in adenoids. Finally, we determined the role of the Mtb T7SS in Mtb entry into adenoid M cells. We infected adenoids with GFP+ WT Mtb or GFP+ Mtb eccD1::Tn and observed more GFP+/EpCAM+/NKM+ cells after infection with WT Mtb as compared to Mtb eccD1::Tn (Figure 5H). Taken together, we conclude that Mtb can enter via mouse NALT and human adenoid M cells in a T7SS dependent manner.

Discussion

In this work, we used in vitro and in vivo M cell models to demonstrate a mucosal interaction between Mtb EsxA and the cell surface protein SR-B1. EsxA, a protein secreted through the T7SS, bound M cells in vitro, was sufficient to mediate M cell translocation by inert beads and was necessary for Mtb translocation in vitro. Furthermore, the T7SS was necessary for Mtb translocation in a mouse mucosal infection model. Primary human airway M cells internalized Mtb in a T7SS-dependent manner, indicating that this process is relevant for human disease. Finally, SR-B1 was enriched on M cells and served as a receptor for Mtb EsxA to mediate Mtb translocation. Together, our data demonstrate a previously undescribed role for Mtb EsxA in mucosal invasion and identify SR-B1 as the airway M cell receptor for Mtb.

EsxA has previously been implicated as a secreted pore-forming molecule (Smith et al., 2008), although this activity has recently been questioned (Conrad et al., 2017). In our experiments utilizing recombinant EsxA, we also did not observe pore formation or epithelial damage. This could be due to the relatively short amount of time we incubated EsxA with our transwells for binding or translocation experiments. Alternatively, the pore forming properties of EsxA may only occur when the protein is in low pH conditions, such as in the lysosome. Thus, EsxA may directly interact with M cell SR-B1 in a cell contact-dependent manner (Conrad et al., 2017), leading to SR-B1 receptor-mediated internalization similar to its function in both hepatitis C virus and Plasmodium vivax uptake (Heo et al., 2006; Manzoni et al., 2017). Although SR-B1 has not been previously identified as an EsxA receptor, prior studies have found other host proteins that interact with EsxA, including laminin (Kinhikar et al., 2010), β2 microglobulin (Sreejit et al., 2014), and TLR-2 (Pathak et al., 2007). We did not identify these proteins in our affinity purification assay, a discrepancy possibly related to the cell types used for binding experiments. However, it is possible that these or other receptors could partly compensate for loss of SR-B1 on M cells, explaining the low level of binding and translocation observed in the absence of SR-B1. Alternatively, Mtb may encode for factors other than EsxA that also mediate M cell binding and translocation, explaining the low level of binding and translocation observed in the absence of EsxA.

In mice, we observed that Mtb lacking the T7SS had a greatly reduced ability to disseminate from mouse NALT to the cervical lymph nodes, potentially due to a reduced ability to translocate across M cells. A possible alternate interpretation for this result centers on the observation that T7SS deficient strains of Mtb are attenuated in vivo and in macrophages (Stanley et al., 2003). Thus, the reduced CFU recovered from draining lymph nodes could represent a macrophage survival defect for the T7SS deficient strains. However, when we used a different attenuated Mtb strain for NALT infection, we observed normal dissemination to the draining lymph nodes. We therefore propose that the reduced CFU recovered from cervical lymph nodes of mice infected with T7SS-deficient Mtb is not simply due to an attenuation defect within macrophages. Consistent with this interpretation, the markedly reduced translocation of T7SS-deficient Mtb across M cells in vitro and into explanted human adenoids ex vivo, in the absence of an innate immune response and over a very short time course, indicates that the T7SS is required for translocation across M cells.

SR-B1 has been well characterized as a high-density lipoprotein receptor involved in cholesterol uptake (Acton et al., 1996). It has also been shown that SR-B1 binds several bacterial molecules, including lipopolysacharide and lipoteicheic acid produced by gram-negative and gram-positive bacteria, respectively (Bocharov et al., 2004). Although direct interaction of EsxA and SR-B1 has not previously been shown, SR-B1 has been reported as a receptor for mycobacteria (Philips et al., 2005; Schäfer et al., 2009), primarily in macrophages (reviewed in Stamm et al., 2015). However, when SR-B1-/- mice were infected with Mtb via the aerosol route, there was no difference in bacterial replication, granuloma size, cytokine secretion, or survival within the first 4 months post-infection compared to wild-type mice (Schäfer et al., 2009). Based on our current data and previous results showing improved mouse survival during aerosol Mtb infection when M cells are reduced (Nair et al., 2016), we predict that loss of M cell SR-B1 should reduce bacterial dissemination from the airway and enhance mouse survival. SR-B1-/- mice experience defective intrauterine and post-natal development and as a result are not born at normal Mendellian ratios (Santander et al., 2013). In addition, they manifest increased serum HDL, cardiovascular defects and altered adrenal hormones (Trigatti et al., 1999), making them incompatible with such a study. Likewise, mice expressing an M-cell specific Cre have not been reported, preventing analysis of SR-B1 function exclusively in M cells.

Adenoid M cells may serve as a portal of entry for Mtb, with significant implications for Mtb pathogenesis in humans. Because respiratory MALT is more abundant in children than adults (Tschernig and Pabst, 2000) and M cells are a key component of MALT (Corr et al., 2008), we propose that the increased incidence of extrapulmonary TB in children (Yang et al., 2004) is due to M cell mediated translocation. Interestingly, there was significant variation in M cell entry in human adenoids, which could relate to polymorphisms in SR-B1 or differences in SR-B1 expression by M cells.

Many bacterial and viral pathogens use the airway as a portal of entry, such as P. aeruginosa (Bucior et al., 2012), Bacillus anthracis (Russell et al., 2008), Streptococcus pneumonia (Wilkosz et al., 2012), Streptococcus pyogenes (Barnett et al., 2015), severe acute respiratory syndrome coronavirus (Liu et al., 2016) and varicella zoster virus (Messaoudi et al., 2009). Similarly, a variety of pathogens that invade via the gastrointestinal tract such as B. abortus via PrP(C) (Nakato et al., 2012), S. typhimurium via GP2 (Hase et al., 2009), and murine norovirus (Gonzalez-Hernandez et al., 2014) possibly via CD300lf (Haga et al., 2016; Orchard et al., 2016) use unique receptors for M cell mediated translocation. Thus, we speculate that a broad array of airway pathogens exploit distinct M cell receptors to penetrate the airway mucosa and disseminate.

In conclusion, we demonstrate that M cells are a portal of entry for Mtb in vitro, in mouse NALT, and in human adenoids. Utilizing mouse models and in vitro models, we identify EsxA and SR-B1 as a molecular synapse required for Mtb translocation across M cells in vitro and in vivo in both mice and humans. A greater understanding of the role of airway M cells in the context of infection by Mtb or other respiratory pathogens will yield insight into novel pathways with potential for new vaccine candidates or therapeutics.

Materials and methods

Bacterial strains and media

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M. tuberculosis Erdman, M. tuberculosis Erdman eccD1::Tn5370 (Cox et al., 1999; Stanley et al., 2003), M. tuberculosis Erdman ΔesxA (Stanley et al., 2003), M. tuberculosis Erdman cor:Tn7 (Zacharia et al., 2013) were grown in Middlebrook 7H9 medium or on Middlebrook 7H11 plates supplemented with 10% oleic acid-albumin-dextrose-catalase. Tween 80 (Fisher T164-500) was added to liquid medium to a final concentration of 0.05%. Strains M. tuberculosis Erdman, M. tuberculosis Erdman eccD1::Tn5370 and M. tuberculosis Erdman ΔesxA underwent whole genome sequencing to determine the presence of unknown genetic polymorphisms.

Whole genome sequencing

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M. tuberculosis Erdman, M. tuberculosis Erdman eccD1::Tn5370, M. tuberculosis Erdman ΔesxA were grown to late-log phase and genomic DNA isolated by the cetyltrimethylammonium bromide (CTAB)-lysozyme method (Larsen et al., 2007). Genomic DNA was then enzymatically fragmented and sequenced using Illumina NextSeq 550 sequencing (Microbial Genome Sequencing Center, https://migscenter.com). To reconstruct the bacterial genomes and identify genetic modifications, the bioinformatics analysis workflow was based on Genome Analysis Toolkit (GATK, v3.8–0; RRID:SCR_001876) (DePristo et al., 2011; McKenna et al., 2010) best practices. Quality control and adapter trimming were performed using Trim Galore (v0.6.4; RRID:SCR_011847) (https://github.com/FelixKrueger/TrimGalore). Burrows-Wheeler Aligner (BWA, v0.7.17; RRID:SCR_010910) (Li and Durbin, 2009) was employed to map the reads to the genome of the publicly available Mtb Erdman (ATCC35801) strain (NCBI assembly: ASM35020v1). Picard (v2.12.0; RRID:SCR_006525) (https://broadinstitute.github.io/picard) was used to remove PCR indices. Variant calling and genotyping were performed using GATK HaplotypeCaller (RRID:SCR_001876) and the variant calls were filtered by applying the following criteria: DP (Approximate read depth)<10, GQ (Genotype Quality)<20. The variants were annotated using a custom Perl script (https://github.com/jiwoongbio/Annomen). Insertions and deletions were identified using coverage depths and split reads from SAMtools (v0.1.19; RRID:SCR_002105) (Li et al., 2009). SPAdes (v3.13.0; RRID:SCR_000131) (Bankevich et al., 2012) was used to de novo assembly and MUMmer 4 (RRID:SCR_001200) (Kurtz et al., 2004) was used to compare the genome assemblies. The genomes are available at NCBI Sequence Read Archive Accession #PRJNA605439.

Cell culture

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The human colorectal adenocarcinoma cell line Caco-2 (HTB-37; RRID:CVCL_0025) and human Burkitt lymphoma cell line Raji B (CCL-86; RRID:CVCL_0511) were obtained from ATCC (Manassas, VA). 16HBE14o- cells (RRID:CVCL_0112) (Forbes et al., 2003) were provided by Dieter Gruenert (University of California, San Francisco). Caco-2 or HBE cells were grown in DMEM (Gibco 11965092) supplemented with 20% fetal bovine serum (Gibco 26140079), 50 units/mL penicillin (Gibco 15140122), 50 μg/mL streptomycin (Gibco 15140122), 2 mM L-glutamine (Gibco 25030081), 1% sodium pyruvate (Gibco 11360070), 1% non-essential amino acids (Gibco 11140050), and 1 mM HEPES (Hyclone SH30237.01). Raji B cells were grown in DMEM supplemented with 20% FBS and 2 mM L-glutamine. In order to generate stable knock-down lines of SR-B1, HBE cells were transduced with lentivirus containing the appropriate shRNA cloned into pLKO.1 (Addgene 10878) as described previously (Huang et al., 2019). Transduced cells were selected with puromycin (Sigma-Aldrich P8833-10MG) and surviving cells were maintained in puromycin for three additional passages. All cell lines were routinely tested for mycoplasma contamination. HBE, Caco-2 and Raji B cell lines were verified using STR profiling analysis (ATCC).

Tissue bilayer model

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3 × 105 Caco-2 or HBE cells in 1 mL of media were plated in the upper chamber of a 3 μm transwell insert (Corning 3462). For Raji B treated transwells, 5 × 105 Raji B cells in 2 mL of media were added to the basal compartment, thereby inducing some of the overlying epithelial cells to differentiate into M cells. For control transwells, 2 mL of media alone were added to the basal chamber, leading to little to no M cell differentiation. 1 mL of media in the upper chamber and 1 mL of media in the bottom chamber were aspirated daily and replaced with 1 mL of fresh media. The transwells were maintained at 37°C for 2 weeks or until the transepithelial electrical resistance was greater than 350 Ω. 72 hr prior to infection, transwells were cultured in media lacking antibiotics. Transwell media was changed approximately 2 hr prior to infection.

In vitro Mtb infection

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Liquid cultures of Mtb were grown until mid-log phase, washed three times with PBS, and centrifuged and sonicated to remove clumps. Bacteria were then resuspended in DMEM + 20% fetal bovine serum. For translocation assays, bacterial inoculum was added to the upper chamber of the transwell at a MOI of 5:1 and media from the basal compartment was sampled after 60 min. The samples were then plated on 7H11 agar plates and maintained in a 37°C incubator for 3 weeks to allow for colony formation.

Protein expression and purification

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gBlocks (IDT) encoding Mtb EsxA or EsxB were first cloned into the pENTR entry vector (Thermo K240020) then subcloned into the pDest17 destination vector (Thermo 11803012; Thermo 11791020) using Gateway cloning (Invitrogen) per the manufacturer’s protocol. The resulting vectors were cloned into the BL21 strain of E. coli (NEB C2527I) for protein expression. 1 L of bacterial culture was grown to an OD600 of 0.6, induced with 1 mM IPTG (Promega V3955) at 37°C for 3 hr, and centrifuged at 3500 rpm for 15 min at 4°C to yield a bacterial pellet. The bacterial pellet was then resuspended in 15 mL of resuspension buffer (50 mM sodium phosphate, 500 mM NaCl, pH 7.4) with one tablet of EDTA-free protease inhibitor (Roche 11836170001). Bacteria were lysed by sonication and centrifuged at 11,200 rpm for 15 min at 4°C. The resulting pellet was resuspended in 20 mL of 8 M urea in resuspension buffer and incubated for 2 hr at room temperature with gentle agitation. The protein slurry was again centrifuged at 11200 rpm for 15 min at 4°C and the resulting supernatant was incubated with cobalt TALON affinity resin (Clontech 635503) for 2 hr at room temperature. Resin was washed with 8 M urea in resuspension buffer and EsxA or EsxB was eluted with 150 mM imidazole and 8 M urea in resuspension buffer. The eluate was dialyzed overnight using a Slide-a-Lyzer dialysis cassette (Thermo 66203) against 10 mM ammonium bicarbonate. The dialyzed sample was again incubated with cobalt TALON affinity resin for 2 hr at room temperature. Resin was subsequently washed with 10 mM Tris-HCl pH 8.0, 0.5% ASB-14 (Sigma A1346-1G) in 10 mM Tris-HCl pH 8.0, and 10 mM Tris-HCl pH 8.0. EsxA or EsxB was eluted with 150 mM imidazole in PBS, dialyzed overnight against PBS, and stored at 4°C. Fractions were analyzed by SDS-PAGE followed by Coommassie staining with Brilliant Blue R-250 (Fisher BP101-25).

Tissue bilayer immunofluorescence microscopy

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In order to image binding of Mtb to transwells, mCherry Mtb was grown until mid-log phase, washed, and centrifuged and sonicated to remove clumps. The bacterial inoculum was added to the upper chamber of the transwell at a MOI of 5:1 for 2 hr at 4°C with gentle agitation every 15 min. Transwells were gently washed and fixed with 4% paraformaldehyde in PBS at 4°C for 1 hr. Transwell inserts were stained with DAPI (Thermo D1306), excised using a blade, mounted on microscope slides using Prolong Gold antifade reagent (Invitrogen P36390) and imaged using an AxioImager MN microscope (Zeiss). In order to image binding of EsxA to transwells, EsxA was expressed and purified as described above. EsxA was then biotinylated by the Sulfo-SBED reagent (Thermo 33033) per manufacturer’s instructions and excess reagent was removed using PD-10 desalting columns (GE Healthcare 17-0851-01). Transwells were then incubated with 1.5 μM EsxA in HBSS for 2 hr at 4°C with gentle agitation, washed, and exposed to UV light for 30 min at room temperature to allow for cross-linking. Transwells were then fixed with 4% paraformaldehyde in PBS for 15 min at room temperature, blocked with 10% donkey serum (Sigma D9663-10ML) in PBS for three hours at room temperature, and incubated with a 1:100 dilution of rabbit anti-SR-B1 antibody (Abcam 52629; RRID:AB_882458) in 2% donkey serum in PBS overnight at 4°C. The following day, transwells were washed and incubated with a 1:100 dilution of PE-conjugated rat NKM 16-2-4 (Miltenyi 130-102-150; RRID:AB_2660295), a 1:500 dilution of an AlexaFluor 647 conjugated donkey-anti-rabbit secondary antibody (Thermo A-31573; RRID:AB_2536183), and a 1:500 dilution of AlexaFluor 488 conjugated streptavidin (Jackson 016-540-084; RRID:AB_2337249) for 1 hr at room temperature. Transwells were then washed, stained with DAPI, excised with a blade, mounted, and imaged as described above. Five fields of view were imaged per independent experiment.

Microsphere conjugation and translocation

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1 µm microspheres were conjugated to protein as per instructions (Polylink 24350–1). Briefly, 12.5 mg of microspheres were centrifuged and washed twice in coupling buffer. Microspheres were then incubated with an EDAC/coupling buffer solution to activate the microspheres. 200 μg of protein is added to the beads, thereby allowing for covalent binding of the protein to the microspheres. Microspheres are then washed twice with PBS and stored at 4°C. In order to test the ability of these beads to translocate in the tissue bilayer assay, beads were diluted to a MOI of 5:1 in DMEM + 20% fetal bovine serum and added to the apical chamber of transwells. Media from the basal compartment was sampled after 60 min and the number of beads present in the sample was analyzed by flow cytometry using an LSR II flow cytometer (BD).

TriCEPS screen

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For initial conjugation of TriCEPS to protein, EsxA or transferrin (300 μg) dissolved in 150 μL 25 mM HEPES pH 8.2 buffer was added to 1.5 μL of the TriCEPS reagent (Dualsystems Biotech) and incubated at 20°C for 90 min with gentle agitation. During this time, 6 × 108 Caco-2 cells were detached from tissue culture plates using 10 mM EDTA in PBS. Cells were split into three aliquots, cooled to 4°C, and pelleted. Each pellet was resuspended in PBS pH 6.5 and sodium metaperiodate was added to a final concentration of 1.5 mM in order to gently oxidize the cell surface. Cells were then incubated with sodium metaperiodate in the dark for 15 min at 4°C. Cells were washed twice with PBS pH 6.5 and split into two new aliquots. TriCEPS coupled EsxA was added to one aliquot and TriCEPS coupled transferrin was added to the other aliquot and incubated for 90 min at 4°C with gentle agitation. Samples were then washed, lysed via sonication, and digested with trypsin. The TriCEPS reagent:ligand:receptor complex was then affinity purified and samples were analyzed using a Thermo LTQ Orbitrap XL spectrometer fitted with an electrospray ion source. Samples were measured in data-dependent acquisition mode in a 90 min gradient using a 10 cm C18 packed column. Samples were analyzed with a statistical ANOVA model with p-values adjusted to control the experiment-wide false discovery rate (FDR). The adjusted p-value obtained for each protein was plotted against the fold enrichment between the two experimental conditions. The area in the volcano plot with an enrichment factor of 4 fold or greater and an FDR-adjusted p-value less than or equal to 0.01 was defined as the receptor candidate space.

Immunoprecipitation

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EsxA was expressed and purified as described above. EsxA or PBS alone was then biotinylated by the Sulfo-SBED reagent (Thermo 33033) according to the manufacturer instructions and excess reagent was removed using PD-10 columns (GE Healthcare 17-0851-01). 1 × 107 HBE cells were detached from tissue culture plates using 10 mM EDTA in PBS. Cells were washed, resuspended in HBSS, and incubated with 1.5 μM EsxA or with PBS alone for 2 hr at 4°C with gentle agitation. Cells were then washed and exposed to UV light for 30 min at room temperature to allow for covalent cross-linking. Cells were lysed with RIPA buffer and lysate was incubated with streptavidin-conjugated magnetic beads (Thermo 88816). Proteins were eluted by boiling and analyzed by SDS-PAGE followed by western blotting with rabbit anti-SR-B1 antibody (Abcam 52629; RRID:AB_882458).

Mouse NALT/human adenoid immunofluorescence

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Mouse NALT sections were obtained as previously described (Nair et al., 2016). Briefly, mouse NALT (after decalcification) and human adenoid specimens were embedded in paraffin, sectioned (5 μm), and mounted on glass slides. Slides were deparaffinized using xylene and ethanol washes followed by heat mediated antigen-retrieval in 10 mM sodium citrate (pH 6.0). Endogenous peroxidase activity was quenched and slides were blocked in 10% donkey serum in PBS for 3 hr at room temperature. Slides were washed with PBS and incubated with a 1:100 dilution of mouse NKM 16-2-4 and rabbit anti-SR-B1 in 2% donkey serum in PBS overnight at 4°C. Slides were then washed with PBS and incubated with a 1:500 dilution of AlexaFluor 568 conjugated goat-anti-mouse secondary antibody (Thermo A-11004; RRID:AB_2534072) or with HRP-conjugated donkey-anti-rabbit secondary antibody (Thermo A16023; RRID:AB_2534697) in 2% donkey serum in PBS for 1 hr at room temperature. Slides were then washed with PBS and incubated with Cy5 tyramide (Perkin Elmer SAT705A001EA) for 8 min. Slides were then washed with PBS, incubated with DAPI, washed with PBS, mounted in Prolong Gold antifade reagent, and imaged using an AxioImager MN microscope (Zeiss). At least three fields of view were imaged per independent experiment.

Mouse intranasal infection

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Mtb Erdman and all mutants were grown in 7H9 and 0.05% Tween-80 until mid-log phase. Cultures were washed three times with PBS, centrifuged to remove clumps, and sonicated to yield a single-cell suspension. Bacteria were resuspended to yield a final concentration of 1 × 108 bacteria in 10 μL PBS. BALB/c mice obtained from The Jackson Laboratory (RRID:IMSR_JAX:000651) were infected with 10 μL of the bacterial suspension intranasally. NALT from three to five mice were collected, homogenized, and plated on 7H11 (Difco 283810) plates supplemented with 10% OADC to enumerate the number of bacteria deposited on Day 0. Mice were sacrificed on Day 7 post-infection and cervical lymph nodes were collected, homogenized, and plated on 7H11 plates. Plates were incubated in a 37°C incubator for 3 weeks to allow for colony formation.

Adenoid culture and infection

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Adenoid samples were obtained from children undergoing elective adenoidectomy for obstructive sleep apnea. Excised adenoids were immediately placed in DMEM, subsequently dissected into 3–4 pieces depending on the size of the adenoid, weighed, and mounted in a 2% agar pad such that only the mucosal surface was exposed. The adenoid pieces were then incubated overnight at 37°C in DMEM supplemented with 20% fetal bovine serum, 2 mM L-glutamine, 1% sodium pyruvate, 1% non-essential amino acids, 1 mM HEPES, 50 ug/mL kanamycin, and 50 ug/mL ampicillin to kill commensal bacteria. The following morning, liquid cultures of GFP Mtb (Kanamycin-resistant) grown to mid-log phase were washed three times with PBS and centrifuged and sonicated to remove clumps. Bacteria were then diluted to 1 × 107 bacteria/mL and 1 mL of inoculum was added to the adenoid and incubated at 37°C for 1 hr. Adenoids were then washed, minced into small pieces, and pushed through a 100 μm nylon cell strainer (Corning 431752). Cells were centrifuged, washed in ACK (Ammonium-Chloride-Potassium) lysis buffer (Gibco A10492-01), and then resuspended in FACS buffer (PBS + 2% FBS). Cells were stained with a 1:100 dilution of mouse anti-EpCAM Brilliant Violet 421 (Biolegend 324219; RRID:AB_11124342), mouse PE-NKM-16-2-4 (Miltenyi 130-102-150; RRID:AB_2660295), or rabbit anti-SR-B1 in FACS buffer, washed, and then incubated with a 1:500 dilution of AlexaFluor 488 conjugated donkey-anti-rabbit secondary antibody (Thermo R37118; RRID:AB_2556546). Cells were washed and fixed in 4% paraformaldehyde for 1 hr followed by counting on an LSRII flow cytometer and analyzed using FlowJo software.

Statistical analysis

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Statistical analysis was performed using GraphPad Prism (RRID:SCR_002798). For in vitro transwell infections to determine bacterial binding or translocation, one-way ANOVA with corrections for multiple comparisons was performed. For in vitro determination of antibody staining, one-way ANOVA with corrections for multiple comparisons was performed. For in vivo adenoid infections or receptor expression, the paired non-parametric Wilcoxon matched pairs signed rank test was performed. For in vivo mouse infections and determination of CFU, the non-parametric Mann-Whitney U test was performed.

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Decision letter

  1. Christina L Stallings
    Reviewing Editor; Washington University School of Medicine, United States
  2. Wendy S Garrett
    Senior Editor; Harvard T.H. Chan School of Public Health, United States
  3. Patricia Champion
    Reviewer

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

Thank you for your detailed response to the reviewers' comments and for submitting this interesting manuscript to eLife! We are excited about the findings you present showing that Mtb EsxA is involved in Mtb invasion of M cells through its engagement with the SR-B1 airway M cell receptor. These findings are an important advance in our understanding of Mycobacterium tuberculosis invasion at different host sites, where the role and mechanisms of M cell invasion is understudied and poorly understood.

Decision letter after peer review:

Thank you for submitting your article "Identification of scavenger receptor B1 as the airway microfold cell receptor for Mycobacterium tuberculosis" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Wendy Garrett as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Patricia Champion (Reviewer #2).

The reviewers have discussed the reviews with one another and the Reviewing Editor. Everyone is very positive about the manuscript and have drafted this decision to help you prepare a revised submission.

Summary:

In this well-presented and interesting manuscript, the authors demonstrate a role for Mtb EsxA in the invasion of M cells through it's engagement with the SR-B1 airway M cell receptor for Mtb. The study is a nice extension of their previous work and an important advance in our understanding of Mycobacterium tuberculosis infection. The reviewers request the following revisions to support the data interpretations made by the authors.

Essential revisions:

1) Please add the following controls to the data shown:

A) Genetic complementation of any of the Mtb strains lacking the T7SS genes.

B) Add EsxB as a control protein for Figure 2 D-H.

C) Add the uninfected control transwell data to Figure 1G.

D) Add a loading control and the EsxA protein pulled down for the western blot in Figure 3B. In addition, are the molecular weight markers correctly indicated for this figure and are there more marker sizes? Unglycosylated SR-BI is indeed ~55-57 kDa, however, this does not seem to be the case in this blot based on the markers. Furthermore, a fully glycosylated SR-BI should be no more than 82 kDa. As such, glycosylation should be verified by EndoH treatment. It is also possible that the band at 130 kDa may represent an SR-BI oligomer, but this should be verified as the estimated molecular weight does not match that of an SR-BI dimer. Have the authors checked to see if that 130 kDa band is actually SR-BI potentially bound to another protein?

2) Please add the following missing information/data:

A) Coomassie gels of the purification of EsxA and EsxB and a discussion of any quality control performed on the proteins, for example MS analysis, to confirm identity/ purity.

B) Methods regarding the LC-MS analysis.

C) For all microscopic images, please indicate how many overall images the presented ones in the manuscript represent. (i.e. How many fields of view were imaged per independent experiment?).

D) Quantification of colocalization of EsxA and SRB-1 in Figure 4.

E) Figure 1B shows approximately 10 bacteria in the field, but the graph in C shows 100-150 area of bacteria per field. Is bacteria area different from the number of bacteria? How were the numbers that are graphed acquired?

F) Figure 2E and 3D state a similar number of nuclei per field was observed, but Figure 2D shows at least two fold higher nuclei than 3C. How are these numbers determined/calculated?

G) For Figure 1B and others, the authors should present the brightfield image to determine where the cell boundaries are to try and distinguish bacteria adherent or inside host cells.

H) In Figure 3, the enrichment of APOE and SRB1 peptides by EsxA IP are significantly less than the enrichment of the transferrin receptor and transferrin. How much or each "receptor" did you pull down relative to the amount of EsxA and transferrin? Is the volcano plot the result of one, two or three independent replicates from the IP?

I) Is it possible to include a schematic of the transwell assay in Figure 1?

J) For Figure 4: The molecular weight of the SR-BI band should be indicated. Is a lower exposure of this immunoblot available?

3) Data files and statistics revisions:

A) The complete proteomics data file supporting the volcano plot in Figure 3A should be submitted.

B) The statistical methods that led to the p-values used to generate the volcano plot in Figure 3A should be added.

C) It is unclear that the Student's t-test is appropriate throughout. For example, I think the t-test is appropriate for comparing treated vs untreated (as RajiB vs Control for a single strain), but inappropriate to go between two strains, or proteins (like 1C-E and 2A, C).

D) Please either designate whether it should be assumed that conditions not called out in the figures are not significantly different from each other, or add a supplemental table of the outcome of all statistical comparisons. For example, would be interesting to know if the deccD +/- RajiB is significantly different from each other in 1C and 1E.

4) Please add the following reference related revisions:

A) The correct reference for SR-BI glycosylation (subsection “Scavenger receptor class B type 1 binds EsxA and is expressed on M cells in vitro”) should be used (older Krieger papers).

B) "Two of the most abundant T7SS secreted proteins are EsxA and EsxB" - Please add reference.

C) Discussion paragraph four – please indicate that Shen et al. is a review. Otherwise, provide the original reference for the identification of SR-BI as an HDL receptor.

Some other points to consider that would strengthen the conclusions made:

1) Something to consider - is performing a direct IP between EsxA and soluble domains of SRB-1 possible? This would sure up the conclusion that SRB1 is an EsxA "receptor" and that the interaction between the two is direct. Alternatively, if the crosslinking assay you are using demonstrates a direct interaction between EsxA and SRB1, despite the fact that the pull down is from lysates, this needs to be clearly stated and discussed.

2) The authors' findings raise the question of whether chemical inhibition of SR-B1 could inhibit binding of EsxA and translocation of Mtb. Using a chemical inhibitor of SR-B1 would alleviate any concerns that genetic knockdown of SR-B1 affects M cell biology more generally. A chemical inhibitor could also be used in vivo.

3) Is it possible to isolate primary M cells from SR-BI-/- mice to definitively identify SR-BI as an EsxA receptor? This would increase the significance of these studies.

4) There are several pieces of data in the manuscript that indicate that EsxA may not be the only factor, or that there may be additional receptors for EsxA on M cells. For example, Figure 4: There are still EsxA+ cells in the absence of SRB1. Also, is translocation into M cells higher for beads with EsxA than Mtb? Does this imply that Mtb surface might include additional factors that modulate this process? Do M. tb without esxA still translocate at a low% , again indicating additional factors? This possibility should be clearly addressed in the Discussion.

https://doi.org/10.7554/eLife.52551.sa1

Author response

Essential revisions:

1) Please add the following controls to the data shown:

A) Genetic complementation of any of the Mtb strains lacking the T7SS genes.

We thank the reviewer for highlighting the need for complementation which has historically been used to control for polar effects or off-target mutations. In our manuscript we chose to use two independent mutants that each have a non-functional T7SS, as it is highly unlikely that both strains would share similar off-target effects of the introduced genetic modifications beyond the known broad impact on the T7SS encoded in the RD1 locus, or that both would have spontaneous mutations in other genomic regions. In addition, both of the strains we used in this study, the MtbΔesxA (Stanley et al., 2007) and Mtb eccD1::Tn5370 (Stanley et al., 2007; Stanley et al., 2003) have been studied previously, including with complementation for T7SS activity (i.e. EsxA secretion), thus excluding polar effects and major genomic changes in the original mutants. To confirm that there were no additional mutations that might have developed during laboratory passage, we sequenced the genomes of all three strains, Mtb Erdman, Mtb ErdmanΔesxA and Mtb Erdman eccD1::Tn5370 and compared them to the published Mtb Erdman reference genome (GenBank: AP012340). The whole genome sequencing identified the expected deletion in esxA along with the presence of the residual transposon sequences and hygromycin cassette used in the generation of the mutant(Mtb esxA in the reference genome is ERDMAN_RS20430 and the deletion is located at NC_020559.1:4333535-4333786) and the transposon insertion in eccD1 (Mtb eccD1 in the reference genome is ERDMAN_RS20440 and the insertion is at NC_020559.1:4337340). As expected, all three strains shared minor differences with the reference genome, and importantly, the Mtb Erdman WT and Mtb eccD1::Tn5370 were identical to each other. When we compared the Mtb Erdman WT to the Mtb ErdmanΔesxA, there was only a single coding variant encoding a missense mutation in Mtb ErdmanΔesxA compared to the Mtb Erdman WT in a putative oxidoreductase. We have uploaded the sequencing data to the Sequence Read Archive (SRA submission # PRJNA605439), and include a variant table in the supplemental material (Supplementary File 1).

B) Add EsxB as a control protein for Figure 2 D-H.

This experiment is now reflected in revised Figure 2D-H.

C) Add the uninfected control transwell data to Figure 1G.

We did not have uninfected data to add, but we did include the translocation data for the control transwells not co-cultured with Raji-B cells in new Figure 1H and Figure 1I.

D) Add a loading control and the EsxA protein pulled down for the western blot in Figure 3B.

We added a loading control as suggested. For the EsxA pulldown, we attempted to detect EsxA after the biotin switch assay but were unable to detect the protein with either the monoclonal EsxA antibody or the anti-His antibody. It is possible that the reaction impacted the epitopes detected by the antibodies.

In addition, are the molecular weight markers correctly indicated for this figure and are there more marker sizes? Unglycosylated SR-BI is indeed ~55-57 kDa, however, this does not seem to be the case in this blot based on the markers. Furthermore, a fully glycosylated SR-BI should be no more than 82 kDa. As such, glycosylation should be verified by EndoH treatment. It is also possible that the band at 130 kDa may represent an SR-BI oligomer, but this should be verified as the estimated molecular weight does not match that of an SR-BI dimer. Have the authors checked to see if that 130 kDa band is actually SR-BI potentially bound to another protein?

We thank the reviewer for identifying the issue regarding the molecular weight of SR-B1. Regarding the molecular weight discrepancy of SR-B1 (130 kDa vs 82 kDa), we returned to our Western blot/IP data and discovered that the reason for the discrepancy was the primary antibody we used to detect SR-B1. In the experiment performed, we used a polyclonal anti-rabbit antibody against SR-B1 (Novus Biologicals NB400-104), which for unclear reasons demonstrated SR-B1 at ~130 kDa both in our hands and on the product website. When we used the mouse monoclonal antibody against SR-B1 that is more commonly used in the field (abcam; ab52629), we demonstrated SR-B1 at the expected molecular weight of 82 kDa (see Author response image 1). We have now redone the biotin switch assay using the ab52629 monoclonal antibody, obtained the appropriate size for SR-B1 and have revised the figure accordingly (New Figure 3B).

Author response image 1

2) Please add the following missing information/data:

A) Coomassie gels of the purification of EsxA and EsxB and a discussion of any quality control performed on the proteins, for example MS analysis, to confirm identity/ purity.

A Coomassie gel is now provided in the supplemental materials (New Figure 2—figure supplement 1).

B) Methods regarding the LC-MS analysis.

Detailed methods for LC-MS are now included in the Materials and methods section.

C) For all microscopic images, please indicate how many overall images the presented ones in the manuscript represent. (i.e. How many fields of view were imaged per independent experiment?).

Depending on the experiment, between 3 to 5 fields per condition were imaged and quantified. This information has been added to the Materials and methods section.

D) Quantification of colocalization of EsxA and SRB-1 in Figure 4.

Quantification is presented in Figure 4F.

E) Figure 1B shows approximately 10 bacteria in the field, but the graph in C shows 100-150 area of bacteria per field. Is bacteria area different from the number of bacteria? How were the numbers that are graphed acquired?

We thank the reviewer for highlighting this confusing point. The image in Figure 1B is a high-power representative image, but not of an entire field. We initially chose to quantify bacteria by using ImageJ to determine the pixel intensity per field as an unbiased surrogate for manually counting bacteria. For clarity, we have changed the analysis so that number of bacteria per 100 cells is quantified in the revised manuscript.

F) Figure 2E and 3D state a similar number of nuclei per field was observed, but Figure 2D shows at least two fold higher nuclei than 3C. How are these numbers determined/calculated?

As above, the images presented are representative images, not of the field in its entirety. Nuclei per field were counted using ImageJ.

G) For Figure 1B and others, the authors should present the brightfield image to determine where the cell boundaries are to try and distinguish bacteria adherent or inside host cells.

While we appreciate the reviewer’s concerns, brightfield images cannot distinguish between adherent and internalized bacteria. To accomplish this would require confocal microscopy with Z-stacks, and such experiments are quite challenging for transwells. As we were interested in binding to the cells, which could include surface binding and subsequently internalized bacteria, we did not pursue additional experimentation.

H) In Figure 3, the enrichment of APOE and SRB1 peptides by EsxA IP are significantly less than the enrichment of the transferrin receptor and transferrin. How much or each "receptor" did you pull down relative to the amount of EsxA and transferrin? Is the volcano plot the result of one, two or three independent replicates from the IP?

The transferrin receptor is a ubiquitously expressed and abundant cell surface protein, which is why the company we contracted, Dualsystems Biotech, provides transferrin as the positive (experimental) control. We only present the transferrin/transferrin receptor data to demonstrate the success of the TriCEPS assay, not for comparative purposes. In addition, it is not possible to quantify the amount of each receptor pulled down using this method. The volcano plot is the result of one experiment with three independent biologic replicates. The latter point is now clearly explained in the figure legend to Figure 3A.

I) Is it possible to include a schematic of the transwell assay in Figure 1?

Added to Figure 1 as new Figure 1B.

J) For Figure 4: The molecular weight of the SR-BI band should be indicated. Is a lower exposure of this immunoblot available?

The molecular weight has now been added and a lower exposure image now replaces Figure 3B.

3) Data files and statistics revisions:

A) The complete proteomics data file supporting the volcano plot in Figure 3A should be submitted.

The complete proteomics data file has been added as supplementary data (new Supplementary File 2).

B) The statistical methods that led to the p-values used to generate the volcano plot in Figure 3A should be added.

Added to the Materials and methods section as suggested.

C) It is unclear that the Student's t-test is appropriate throughout. For example, I think the t-test is appropriate for comparing treated vs untreated (as RajiB vs Control for a single strain), but inappropriate to go between two strains, or proteins (like 1C-E and 2A, C).

For the entire manuscript, we replaced Student’s t-test by one-way ANOVA with corrections for multiple comparisons as it is a more rigorous statistical analysis tool. The Materials and methods and figure legends have been updated with this change.

D) Please either designate whether it should be assumed that conditions not called out in the figures are not significantly different from each other, or add a supplemental table of the outcome of all statistical comparisons. For example, would be interesting to know if the deccD +/- RajiB is significantly different from each other in 1C and 1E.

We have added a comment to the legend for each figure where appropriate indicating that if comparisons are not directly highlighted, that they are not statistically significant.

4) Please add the following reference related revisions:

A) The correct reference for SR-BI glycosylation (subsection “Scavenger receptor class B type 1 binds EsxA and is expressed on M cells in vitro”) should be used (older Krieger papers).

Thank you for the suggestion. We revised this section to reflect the new data (see comment 1D above) and include the original paper by Acton et al. to indicate the correct MW.

B) "Two of the most abundant T7SS secreted proteins are EsxA and EsxB" - Please add reference.

Thank you for the suggestion. We have included references detailing the abundance of these two proteins.

C) Discussion paragraph four - please indicate that Shen et al. is a review. Otherwise, provide the original reference for the identification of SR-BI as an HDL receptor.

We changed the reference to indicate the original identification of SR-B1 as an HDL receptor.

Some other points to consider that would strengthen the conclusions made:

1) Something to consider - is performing a direct IP between EsxA and soluble domains of SRB-1 possible? This would sure up the conclusion that SRB1 is an EsxA "receptor" and that the interaction between the two is direct. Alternatively, if the crosslinking assay you are using demonstrates a direct interaction between EsxA and SRB1, despite the fact that the pull down is from lysates, this needs to be clearly stated and discussed.

Thank you for the suggestion. Performing an IP of EsxA with a soluble SR-B1 domain would be a challenge as it is not known if the three-dimensional structures of soluble (i.e. recombinant) and transmembrane SR-B1 are the same, and thus soluble SR-B1 may not bind EsxA for technical rather than biologic reasons. We performed two independent assays using EsxA to probe for cell surface binding. While both included a cross-linking step (TriCEPs and biotin switch), they were also both performed on whole cells, not lysates, and both demonstrated an interaction between EsxA and SR-B1.

2) The authors' findings raise the question of whether chemical inhibition of SR-B1 could inhibit binding of EsxA and translocation of Mtb. Using a chemical inhibitor of SR-B1 would alleviate any concerns that genetic knockdown of SR-B1 affects M cell biology more generally. A chemical inhibitor could also be used in vivo.

Thank you for the suggestion. While we recognize this is an important experiment, we are concerned that an inhibitor of SR-B1 activity that blocks HDL internalization (such as the established HDL internalization inhibitor BLT-1) might not share the same inhibitory activity for Mtb binding/internalization. As such, only a positive result (i.e. an inhibitor successfully prevents Mtb internalization) would be interpretable. In addition, administration of such an inhibitor in vivo could yield confounding results because it could not only impact M cell internalization but also macrophage SR-B1 mediated internalization.

3) Is it possible to isolate primary M cells from SR-BI-/- mice to definitively identify SR-BI as an EsxA receptor? This would increase the significance of these studies.

SR-B1-/- mice are nearly impossible to breed and have multiple developmental and physiologic abnormalities. Ongoing work in the lab is focused on developing a M cell specific Cre mouse that will allow for cell type specific conditional deletion of SR-B1 from M cells by crossing to an SR-B1fl/fl mouse. However, validating and testing Cre expression, crossing these mice and then testing them for M cell activity is beyond the scope of this manuscript.

4) There are several pieces of data in the manuscript that indicate that EsxA may not be the only factor, or that there may be additional receptors for EsxA on M cells. For example, Figure 4: There are still EsxA+ cells in the absence of SRB1. Also, is translocation into M cells higher for beads with EsxA than Mtb? Does this imply that Mtb surface might include additional factors that modulate this process? Do M. tb without esxA still translocate at a low% , again indicating additional factors? This possibility should be clearly addressed in the Discussion.

Thank you for raising these important points. We have expanded the Discussion to incorporate the idea that there may be additional EsxA receptors on M cells, and that other Mtb factors may also mediate M cell entry.

References:

Stanley, SA, Johndrow, JE, Manzanillo, P, Cox, JS.2007. The Type I IFN response to infection with Mycobacterium tuberculosis requires ESX-1-mediated secretion and contributes to pathogenesis. J Immunol 178:3143–3152.

https://doi.org/10.7554/eLife.52551.sa2

Article and author information

Author details

  1. Haaris S Khan

    Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, United States
    Contribution
    Conceptualization, Formal analysis, Investigation, Visualization, Writing - original draft, Project administration, Writing - review and editing
    Competing interests
    No competing interests declared
  2. Vidhya R Nair

    Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, United States
    Contribution
    Conceptualization, Formal analysis, Investigation, Writing - review and editing
    Competing interests
    No competing interests declared
  3. Cody R Ruhl

    Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, United States
    Contribution
    Investigation, Writing - review and editing
    Competing interests
    No competing interests declared
  4. Samuel Alvarez-Arguedas

    Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, United States
    Contribution
    Investigation, Writing - review and editing
    Competing interests
    No competing interests declared
  5. Jorge L Galvan Rendiz

    Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, United States
    Contribution
    Investigation, Writing - review and editing
    Competing interests
    No competing interests declared
  6. Luis H Franco

    Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, United States
    Present address
    Federal University of Minas Gerais, Belo Horizonte, Brazil
    Contribution
    Investigation, Writing - review and editing
    Competing interests
    No competing interests declared
  7. Linzhang Huang

    Center for Pulmonary and Vascular Biology, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, United States
    Contribution
    Resources, Writing - review and editing
    Competing interests
    No competing interests declared
  8. Philip W Shaul

    Center for Pulmonary and Vascular Biology, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, United States
    Contribution
    Resources, Funding acquisition, Writing - review and editing
    Competing interests
    No competing interests declared
  9. Jiwoong Kim

    Quantitative Biomedical Research Center, Department of Population and Data Sciences, University of Texas Southwestern Medical Center, Dallas, United States
    Contribution
    Data curation, Software, Formal analysis, Writing - review and editing
    Competing interests
    No competing interests declared
  10. Yang Xie

    1. Quantitative Biomedical Research Center, Department of Population and Data Sciences, University of Texas Southwestern Medical Center, Dallas, United States
    2. Harold C Simmons Cancer Center, University of Texas Southwestern Medical Center, Dallas, United States
    3. Department of Bioinformatics, University of Texas Southwestern Medical Center, Dallas, United States
    Contribution
    Resources, Software, Supervision, Writing - review and editing
    Competing interests
    No competing interests declared
  11. Ron B Mitchell

    Department of Otolaryngology, University of Texas Southwestern Medical Center, Dallas, United States
    Contribution
    Resources, Writing - review and editing
    Competing interests
    No competing interests declared
  12. Michael U Shiloh

    1. Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, United States
    2. Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, United States
    Contribution
    Conceptualization, Formal analysis, Supervision, Funding acquisition, Visualization, Writing - original draft, Project administration, Writing - review and editing
    For correspondence
    michael.shiloh@utsouthwestern.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4329-2253

Funding

National Institute of Allergy and Infectious Diseases (AI125939)

  • Michael U Shiloh

National Institute of Allergy and Infectious Diseases (AI142784)

  • Michael U Shiloh

National Institute of Allergy and Infectious Diseases (5T32AI005284)

  • Haaris S Khan

National Heart, Lung, and Blood Institute (HK131597)

  • Philip W Shaul

Burroughs Wellcome Fund (1017894)

  • Michael U Shiloh

Welch Foundation (I-1964-20180324)

  • Michael U Shiloh

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Beth Levine and members of the Shiloh Lab for constructive feedback on the manuscript. This work is supported by the Burroughs Wellcome Fund 1017894 (MUS), Welch Foundation I-1964–20180324 (MUS), NIH U01 AI125939-04 (MUS), NIH U19 AI142784-01 (MUS), NIH 5T32AI005284-40 (HSK), and NIH R01 HL131597-03 (PWS).

Ethics

Human subjects: Human adenoids were obtained from children undergoing elective adenoidectomy for sleep apnea after informed consent was obtained from parents or guardians. This study was reviewed by the University of Texas Southwestern Institutional Review Board (protocol STU 062016-087).

Animal experimentation: Animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee at the University of Texas Southwestern (protocol 2017-101836) and followed the eighth edition of the Guide for the Care and Use of Laboratory Animals. The University of Texas Southwestern is accredited by the American Association for Accreditation of Laboratory Animal Care (AAALAC).

Senior Editor

  1. Wendy S Garrett, Harvard T.H. Chan School of Public Health, United States

Reviewing Editor

  1. Christina L Stallings, Washington University School of Medicine, United States

Reviewer

  1. Patricia Champion

Publication history

  1. Received: October 8, 2019
  2. Accepted: March 4, 2020
  3. Accepted Manuscript published: March 5, 2020 (version 1)
  4. Version of Record published: March 11, 2020 (version 2)
  5. Version of Record updated: March 11, 2020 (version 3)

Copyright

© 2020, Khan et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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