HN878 Infection in MicetuberculosisMycobacteriumCXCL17 Is Dispensable during Hypervirulent

Marcela Muñoz-Torrico, Silvia Guzmán-Beltrán, Joaquín Zúñiga and Shabaana A. KhaderJosé Alberto Choreño-Parra, Micah D. Dunlap, Rosemary Swanson, Luis A. Jiménez-Álvarez,

http://www.immunohorizons.org/content/5/9/752https://doi.org/10.4049/immunohorizons.2100048doi:

2021, 5 (9) 752-759ImmunoHorizons 

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CXCL17 Is Dispensable during Hypervirulent Mycobacteriumtuberculosis HN878 Infection in Mice

Jos�e Alberto Chore~no-Parra,*,†,‡ Micah D. Dunlap,‡ Rosemary Swanson,‡ Luis A. Jim�enez-�Alvarez,*Marcela Mu~noz-Torrico,§ Silvia Guzm�an-Beltr�an,{ Joaquín Z�u~niga,*,|| and Shabaana A. Khader‡

*Instituto Nacional de Enfermedades Respiratorias Ismael Cosío Villegas, Mexico City, Mexico; †Escuela Nacional de Ciencias Bil�ogicas, Instituto Polit�ecnicoNacional, Mexico City, Mexico; ‡Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO; §Clinica de Tuberculosis,Instituto Nacional de Enfermedades Respiratorias Ismael Cosío Villegas, Mexico City, Mexico; {Investigaci�on en Microbiología, Instituto Nacional deEnfermedades Respiratorias Ismael Cosío Villegas, Mexico City, Mexico; and ||Tecnologico de Monterrey, Escuela de Medicina y Ciencias Biom�edicas, MexicoCity, Mexico

ABSTRACT

CXCL17 is a novel mucosal chemokine that mediates myeloid cell recruitment and bactericidal activity and highly expressed in the

respiratory tract. However, its role in tuberculosis (TB) immunopathogenesis or protection remains unknown. In this study, we

evaluated the function of CXCL17 in a mouse model of aerosol infection with the clinical W-Beijing lineageMycobacterium

tuberculosis hypervirulent HN878 strain. Our results show that CXCL17 production increases in the lung of M. tuberculosis–infected

mice during acute and chronic stages of infection. Moreover, in vitroM. tuberculosis infection of epithelial cells and myeloid cells

induces production of CXCL17. In humans, lower serum CXCL17 levels are observed among active pulmonary TB patients when

compared with subjects with latent TB infection and healthy controls, suggesting a protective role. However, mice treated with

rCXCL17 show similar lung bacterial burden and inflammation compared with control animals, despite an increased lung myeloid cell

accumulation. Finally, CXCL17�/� mice are not more susceptible to TB than wild-type animals. These findings suggest that CXCL17 is

induced in both murine epithelial and myeloid cells uponM. tuberculosis infection and increased expression during human latent TB

infection. However, CXCL17 may have a dispensable role during pulmonary TB. ImmunoHorizons, 2021, 5: 752–759.

INTRODUCTION

Mycobacterium tuberculosis, the causative agent of tuberculo-sis (TB), is a leading infectious cause of death worldwide.Host immunity against M. tuberculosis requires a balanced

interaction of different chemokine/chemokine receptor axescontrolling leukocyte recruitment into the lung (1). Blockingpathological chemokine-mediated activities or harnessing ben-eficial chemokine/receptor axes would constitute critical ther-apeutic strategies for TB. This emphasizes the importance of

Received for publication May 24, 2021. Accepted for publication September 3, 2021.

Address correspondence and reprint requests to: Dr. Shabaana A. Khader, Department of Molecular Microbiology, Washington University School of Medicine, St.Louis, Campus Box 8230, 660 S. Euclid Avenue, St. Louis, MO 63110. E-mail address: sakhader@wustl.edu

ORCIDs: 0000-0002-5326-2720 (L.A.J.-A.); 0000-0002-7143-0281 (J.Z.); 0000-0002-9545-4982 (S.A.K.).

This work was supported by the National Council on Science and Technology of Mexico (CONACyT–CVU-737347 to J.A.C-.P. to achieve his Ph.D. degree). This workwas also supported by the Washington University School of Medicine and National Institutes of Health Grants HL105427, AI111914-02, AI134236-02, and AI123780 (toS.A.K.).

Author contributions: J.A.C.-P. participated in the intellectual conception of the study, did the experiments with mice, analyzed human and mouse data, and draftedthe manuscript. M.D.D. and R.S. contributed to the experiments with mice and curated the text of the manuscript. L.A.J.-A. and M.M.-T. collected human serumsamples and measured CXCL17 levels. J.A.C.-P. and S.G.-B. performed killing assays withMycobacterium tuberculosis HN878 and H37Rv. J.Z. and S.A.K. designed thestudy, contributed to the writing process of the manuscript, and revised it for intellectual content. All the authors read and approved the final version of themanuscript.

Abbreviations used in this article: AM, alveolar macrophage; B6, C57BL/6; BMDC, bone marrow–derived DC; BMDM, bone marrow–derived macrophage; DC,dendritic cell; dpi, day post–M. tuberculosis HN878 infection; GPR35, G protein–coupled receptor 3; HC, healthy control; LTBI, latent TB-infected; mDC, myeloiddendritic cell; PTB, pulmonary TB; RM, recruited macrophage; TB, tuberculosis; WT, wild-type.

This article is distributed under the terms of the CC BY 4.0 Unported license.

Copyright © 2021 The Authors

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RESEARCH ARTICLE

Infectious Disease

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a complete understanding of the role of chemokines duringTB, especially of those that are yet to be assigned a specificfunction.

CXCL17 is a novel mucosal chemotactic factor for myeloidcells that also possesses a broad spectrum of bactericidal activ-ity (2�4). This chemokine is expressed along the respiratoryepithelium at a steady state and under inflammatory conditions(2, 3, 5), making it a potential participant in mediating lungdefense against pathogens. CXCL17 induces rigorous migrationof monocytes and dendritic cells (DCs) via ERK1/2 and p38MAPK signaling (2, 6). These chemotactic properties might beof special relevance during TB because the myeloid cell infiltra-tion into the lung is a crucial determinant of the protective ver-sus pathogenic nature of tubercle granulomas. For instance, thepresence of CCR21 alveolar macrophages (AMs) in the airwayis pivotal for the initiation of anti�M. tuberculosis immunity.Upon engulfing bacilli, these cells migrate to the lung paren-chyma and promote the formation of protective granulomas (7).Conversely, interstitial IFN-responsive macrophages and plas-mocytoid DCs might be more permissive for M. tuberculosisgrowth or induce inflammatory reactions that result in lung tis-sue damage during active TB (8).

Interestingly, healthy CXCL17�/� mice have decreasednumbers of AMs but an intact number of interstitial lung mac-rophages at the steady state (4). Therefore, CXCL17 may alsoparticipate in the early regulation of the myeloid cell composi-tion of TB granulomas. We previously showed overexpressionof the CXCL17 gene among innate lymphoid cells isolated fromthe lung of TB patients (9). However, the role of CXCL17 inprotective immunity against M. tuberculosis remains elusive. Inthe current study, we addressed whether CXCL17 participatesin immune protection against TB in a model of hypervirulentM. tuberculosis HN878 infection in mice. Our results indicatethat murine M. tuberculosis HN878 infection induces CXCL17production within the lung and in both epithelial and myeloidcells after early and late time points following infection. Fur-thermore, CXCL17 treatment attracts monocytes to the site ofM. tuberculosis HN878 infection. In M. tuberculosis�infectedindividuals, serum levels of CXCL17 are higher among latentTB-infected (LTBI) subjects than individuals with active pul-monary TB (PTB), suggesting a protective role. However,CXCL17�/� mice are not more susceptible to M. tuberculosisHN878 infection than wild-type (WT) controls. These findingssuggest that CXCL17 is induced in murine epithelial and mye-loid cells upon M. tuberculosis exposure, and increased levelsaccumulate in human LTBI. However, CXCL17 expression isdispensable following hypervirulent M. tuberculosis HN878infection in mice.

MATERIALS AND METHODS

Human samplesPeripheral blood samples were obtained from individuals withactive PTB and LTBI that were recruited at the TB clinic of

the Instituto Nacional de Enfermedades Respiratorias IsmaelCosío Villegas in Mexico City, Mexico. Subjects with immuno-deficiencies, patients with diabetes, and those taking anti-TBdrugs before recruitment were not eligible. A group of healthyindividuals was also included and served as healthy controls(HC). Clinical and demographic data from participants wereobtained by direct clinical interview, physical examination, andreview of their medical records. PTB patients were evaluatedby chest x-ray images, and the degree of lung damage wasdetermined using a quantitative scale, as previously described(10, 11). Briefly, lung parenchyma was divided in four quad-rants, with the division between the bilateral upper and lowerlung set at the carina section. Each quadrant was scored from0�5, where 0 denoted absence of lesions and 5 denoted exten-sive effect. All patients provided written informed consent toparticipate in the study. The Institutional Review Board of theInstituto Nacional de Enfermedades Respiratorias Ismael CosíoVillegas approved the study.

MiceWT C57BL/6 (B6) mice were purchased from The JacksonLaboratory (Bar Harbor, ME). Cxcl17tm1b knockout mice(CXCL17�/�) were obtained from the Knockout Mouse Reposi-tory (University of California Davis, Davis, CA). Animals werebred at the animal facility in the Washington University Schoolof Medicine (St Louis, MO). Experimental mice were age andsex matched and used between the ages of 6�8 wk, in accor-dance with the Institutional Animal Care and Use Committeeguidelines at Washington University, and approved under pro-tocol 20160129.

M. tuberculosis infection and CXCL17 treatmentM. tuberculosis HN878 (BEI Resources, Manassas, VA; NationalInstitutes of Health contract AI-75320) and M. tuberculosisH37Rv were cultured in Proskauer�Beck medium containing0.05% Tween 80 to reach midlog phase and frozen in 1-ml ali-quots at �80�C until used. Mice were aerosol-infected with�100 CFUs of M. tuberculosis HN878 using a Glas-Col airborneinfection system and euthanized at given time points. Some B6mice received �100 mg/kg mouse rCXCL17 (4270-DM/CF; R&DSystems, Minneapolis, MN) administered intratracheally in 50 mlof sterile PBS per mouse at different days post�M. tuberculosisHN878 infection (dpi). Pulmonary bacterial burden was deter-mined by plating 10-fold serial dilutions of lung homogenates onPetri dishes containing 7H11 agar solid medium and countingCFUs.

In vitro infectionsTotal lung cell suspensions, C10 epithelial cells, bone mar-row�derived macrophages (BMDMs), and bone marrow�derivedDCs (BMDCs) were obtained and cultured as described before(7). Cells were infected with M. tuberculosis HN878 at a multi-plicity of infection of 1 in antibiotic-free complete DMEM, andculture supernatants were collected after 48 h.

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HistologyMouse lungs were inflated with 10% neutral buffered formalinand embedded in paraffin. Five-micrometer lung sections werestained with H&E and processed for light microscopy. For cal-culation area of inflamed lung sections, representative imageswere taken with the Hamamatsu NanoZoomer 2.0-HT systemwith NDP scan image acquisition software. The percentagearea occupied by inflammation was calculated as describedbefore (12).

Flow cytometryAt given dpi, lung single-cell suspensions were obtained andtreated with Fc Block (CD16/CD32,2.4G2; Tonbo Biosciences,San Diego, CA) as previously described (7). Lung cells wereincubated with fluorochrome-labeled specific Abs: CD11c (HL3;BD Biosciences), CD11b (M1/70; BD Biosciences and TonboBiosciences), SiglecF (E50�2440; BD Biosciences), and Gr-1(RB6�8C5; BD Biosciences). Cells were acquired using a BDLSR Fortessa flow cytometer and gated based on their forwardand side scatter characteristics. Neutrophils, monocytes,recruited macrophages (RMs), myeloid DCs (mDCs), and AMswere defined as before (7). The frequency of specific cell typeswas calculated using FlowJo (Flow Jo, Ashland, OR).

CXCL17 quantificationMouse and human CXCL17 levels in lung homogenates, culturesupernatants, and serum samples were determined by ELISAusing commercial kits (MBS916471 and MBS7232792; MyBio-Source, San Diego, CA).

Lung cytokine expressionTotal lung RNA was extracted using the RNeasy Mini Kit(QIAGEN), and DNase I (QIAGEN)�treated cDNA was gener-ated using Applied Biosystems (ABI) reverse transcriptionreagents (ABI/Thermo Fisher Scientific) and then amplifiedwith FAM-tagged probes and real-time PCR primers on aViiA7 Real-Time PCR System (Life Technologies/ThermoFisher Scientific). Specific gene expression was calculated rela-tive to GAPDH mRNA expression. Primer and probe sequencestargeting specific genes (IL-1b, TNF-a, IFN-g, IL-17, and IL-10) were commercially purchased (ABI).

Bactericidal assayA stock of M. tuberculosis HN878 and H37Rv was thawed, and500 ml was transferred to a sterile glass tube containing Pros-kauer�Beck medium. The cultures were incubated at 37�C,7.5% CO2, with continuous shaking (100 rpm) for 7 d to allowbacteria to reach midlog phase. Afterward, 1ml from each cul-ture was taken and diluted (1:75 for M. tuberculosis HN878,1:50 for M. tuberculosis H37Rv) in Proskauer�Beck medium.The bacterial suspension was placed in a 96-well plate by add-ing 100 ml/well. An extra 100 ml of human rCXCL17 (0.5mM,1mM, and 2mM) was added to the corresponding wells (finalvolume of reaction 200 ml/well) using saline solution and

rifampicin (5mg/ml) as negative and positive controls, respec-tively. Treated bacteria were incubated at 37�C, 7.5% CO2, for 5d, then 10-fold serial dilutions of the cultures were plated onPetri dishes containing 7H11 agar solid medium, and CFUswere determined after 14�21 d. Each condition was performedin triplicate.

Statistical analysisStatistical analyses were performed using GraphPad Prism 5(GraphPad, La Jolla, CA). Specific analysis tests are mentioned inthe figure legends. The p values # 0.05 were considered signifi-cant (*p # 0.05, **p # 0.01, ***p # 0.001, and ****p # 0.0001).

RESULTS

M. tuberculosisHN878 infection-induced CXCL17production in mouse lungsDifferent microbial stimuli are known to induce CXCL17expression (4, 6, 13). To address whether CXCL17 is inducedduring M. tuberculosis infection, B6 mice were infected withthe clinical W-Beijing lineage M. tuberculosis hypervirulentstrain HN878, and CXCL17 levels were determined in lunghomogenates. We observed increased CXCL17 production inthe lung of infected animals at both acute and chronic stages ofTB when compared with levels measured in uninfected animals(Fig. 1A). Using total lung cell suspensions from B6 mice, wealso found that the in vitro exposure to M. tuberculosis HN878induced increased CXCL17 production in lung cells (Fig. 1B).To further evaluate possible cellular sources of this chemokineduring TB, mouse lung epithelial cells, BMDMs, and BMDCswere exposed to M. tuberculosis HN878 in vitro. Indeed, all cellsubtypes produced high amounts of CXCL17 after the bacterialexposure (Fig. 1B), suggesting that both epithelial and myeloidcellular sources produce CXCL17 in response to M. tuberculosisinfection.

To address whether CXCL17 was induced in individualsinfected with M. tuberculosis, we measured CXCL17 levelsin serum collected from LTBI and PTB patients. We did notobserve an increase in serum levels of CXCL17 in humanswith either LTBI or PTB compared with HC (Fig. 1C).Indeed, PTB patients showed a significant decrease inserum CXCL17 amounts when compared with LTBI andHC, whereas no differences between HC and LTBI wereobserved. Additionally, CXCL17 levels did not correlatewith increased lung damage as measured by a validatedradiological score among PTB patients (Fig. 1D). These datasuggest that, whereas CXCL17 expression is induced inlungs during TB in mice, expression in the peripheral bloodis decreased in individuals with PTB.

CXCL17 expression is dispensable for host immunity in themouse modelTo better characterize a functional role for CXCL17, we usedthe mouse model of hypervirulent M. tuberculosis infection. We

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first tested the effect of the exogenous rCXCL17 treatment onM. tuberculosis HN878 infection. Two groups of B6 mice wereinfected with M. tuberculosis HN878; one group was treatedwith rCXCL17 at 0, 7, and 14 dpi, and the other received PBS.At 30 dpi, we measured immune cell recruitment and foundthat M. tuberculosis HN878-infected mice treated withrCXCL17 showed increased recruitment of mDCs, monocytes,and RMs in the infected lung when compared with PBS-receiv-ing control M. tuberculosis HN878-infected lungs (Fig. 2A). Nodifferences in the number of neutrophils and AMs wereobserved between animal groups (Fig. 2A). In contrast, lungbacterial burden and lung inflammation measured was similarbetween CXCL17-treated and control M. tuberculosis HN878-infected lungs (Fig. 2B�D).

Given the possibility that the dosage, half-life, and timing ofrCXCL17 administration was inadequate to observe a strong effecton M. tuberculosis CFU at 30 dpi, we then analyzed anothergroup of mice infected with M. tuberculosis HN878 and treatedwith rCXCL17 but sacrificed 48 h after the last dose (16 dpi).Again, no differences in lung bacterial burden were observed ascompared with mice that received PBS (Fig. 2E). Additionally,rCXCL17 treatment did not impact expression of cytokines intotal lung tissue, as we did not find any differences in the expres-sion of IL-1b, TNF-a, IFN-g, IL-17, and IL-10 between M.

tuberculosis HN878-infected B6 mice receiving rCXCL17 whencompared with M. tuberculosis�infected mice treated with PBS(Fig. 2F). Together, these data demonstrate that increased signal-ing through the CXCL17/CXCL17�receptor axis improved mye-loid cell recruitment to the lungs of B6 mice without improvingoverall M. tuberculosis control and lung tissue inflammation.

Finally, we evaluated immune protection against TB in theabsence of CXCL17 signaling. WT and CXCL17�/� mice wereinfected with M. tuberculosis HN878 as before. After 30 dpi,CXCL17�/� showed impaired recruitment of monocytes andRMs to the lung, with no differences in the number of mDCs,AMs, and neutrophils with respect to WT mice (Fig. 3A). How-ever, CXCL17�/� mice showed similar levels of lung bacterialburden at 30, 100, and 150 dpi compared with WT controls (Fig.3B). Furthermore, no differences in lung inflammation occurredbetween animal groups at 100 dpi (Fig. 3C, 3D). Hence, our dataindicate that, whereas CXCL17 is induced in the murine lungduring PTB, CXCL17 expression is not required for protectiveimmunity against M. tuberculosis HN878 infection in mice.

CXCL17 lacks bactericidal activity againstM. tuberculosisAn important property of CXCL17 is that this chemokine candisrupt the membrane of Escherichia coli, Staphylococcus aureus,

FIGURE 1. Induction of CXCL17 in mice and humans with TB.

(A) B6 mice were infected with �100 CFU of aerosolizedM. tuberculosis HN878, and CXCL17 levels were measured at 30 and 100 dpi in lung homo-

genates. The CXCL17 levels in M. tuberculosis–infected mice were compared with basal levels of chemokine production in lung homogenates from

uninfected animals (n 5 5 mice per time point) using the Kruskal–Wallis test and the post hoc Dunn test for multiple comparisons. (B) Total lung cell

suspensions, C-10 mouse lung epithelial cells, BMDMs, and BMDCs, were infected with M. tuberculosis HN878 at a multiplicity of infection 51 for

48 h. Levels of CXCL17 in supernatants from infected and uninfected cells were compared using the unpaired Student t test (n 5 5 per group). The

data shown represent mean (6 SD) values from two to three independent experiments per time point and experimental condition. (C) Levels of

CXCL17 in serum from HC (n 5 16), subjects with LTBI (n 5 25), and patients with active PTB (n 5 47) were compared using the Kruskal–Wallis test

and the post hoc Dunn multiple comparisons test. (D) The correlation between serum levels of CXCL17 and values obtained in a radiological lung

damage score in PTB patients was analyzed using the Pearson correlation coefficient (n 5 47). *p # 0.05, **p # 0.01, ****p # 0.0001.

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Salmonella sp., Pseudomonas aeruginosa, and Candida albicans(3). To address a lytic effect on M. tuberculosis, we cultured M.tuberculosis HN878, and the laboratory adapted M. tuberculosisstrain H37Rv in presence of CXCL17. For this purpose, weused 0.5 mM, 1 mM, and 2 mM human rCXCL17, concentrationsat which this chemokine shows an important killing activityagainst other pathogens (3). Of note, human rCXCL17 showedno effect on M. tuberculosis HN878 growth at any concentra-tion (Fig. 4A) compared with rifampicin, whereas only a mod-est reduction of the survival of M. tuberculosis H37Rv wasobserved at 2 mM (Fig. 4B). Hence, these findings indicate thatCXCL17 lacks direct lytic capacity to kill M. tuberculosis bacilli.

DISCUSSION

Several chemokine/chemokine receptor axes are essential forimmune protection against M. tuberculosis (1). Some mediatebeneficial activities, such as the adequate localization of differ-ent leukocyte subtypes at the site of M. tuberculosis infection.

For instance, CXCL13 promotes the recruitment of CXCR51

follicular B- and T helper cells into the M. tuberculosis�infectedlung, where they form ectopic lymphoid follicles that supportprotective immunity against TB (14). Similarly, CCR2 signalingaids AMs in exiting airways and migrating to lung granulomas,and the absence of CCR2 leads to increased susceptibility to TB(7). Conversely, other chemotactic factors (i.e., S100A8/A9)mediate the accumulation of neutrophils into granulomas, exac-erbating inflammation (15). Despite these advances, novel che-motactic factors of unknown function during TB have beenrecently discovered. This is the case for CXCL17, a novel muco-sal chemokine constitutively produced along the respiratorytract (2, 3, 5), whose expression is further potentiated byinflammatory and microbial stimuli (4, 6). CXCL17 is a potentialparticipant in the immunity of the lung. However, little litera-ture exists about its implication during respiratory infections.Thus, our study is among the first to address the role ofCXCL17 in immunity against a relevant respiratory pathogensuch as M. tuberculosis.

FIGURE 2. Administration of rCXCL17 does

not improve the control of M. tuberculosis

HN878 infection in mice.

B6 mice were infected with �100 CFU of

aerosolized M. tuberculosis HN878 as before

and sacrificed at 30 dpi. Some mice (n 5 5)

received �100mg/kg rCXCL17 administered

by the intratracheal route at 0, 7, and 14 dpi,

whereas a separate group of animals (n 5 10)

were treated with PBS. (A) Lung myeloid cell

populations were enumerated in M. tubercu-

losis HN878-infected B6 mice treated with

rCXCL17 and control animals using flow

cytometry. (B) Lung bacterial burden was

determined by plating and counting CFUs. (C)

Lung area occupied by inflammation was

quantified in H&E-stained, formalin-fixed, and

paraffin-embedded (FFPE) lungs using Nano-

Zoomer software. (D) Representative micro-

photographs showing inflammation in H&E-

stained FFPE lung sections (original magnifi-

cation �10) from rCXCL17-treated and

untreated M. tuberculosis HN878-infected B6

mice. (E) Another group of B6 mice infected

with M. tuberculosis HN878 was treated with

rCXCL17 and sacrificed at 16 dpi. Lung bacte-

rial burden was determined by plating. (F) The

expression of several cytokines in the lungs of

mice was assessed by real-time PCR. Com-

parisons between groups were performed

using the unpaired Student t test. The data

shown represent mean (6 SD) values from

two to three independent experiments. *p #

0.05, **p # 0.01.

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CXCL17 mediates the recruitment of myeloid cells to thelung (4). Among cell subtypes responding to CXCL17 are DCs,monocytes, and macrophages (2, 4, 6). In this study, we con-firmed these chemotactic properties of CXCL17, as we foundthat intratracheal administration of rCXCL17 to M. tuberculosisHN878-infected mice increased the accumulation of mDCs,monocytes, and RMs into the lung. Moreover, M. tuberculo-sis�infected CXCL17�/� mice cannot recruit monocytes and

RMs but have no deficiencies in lung mDCs. In this regard, ithas been proposed that the activity of CXCL17 is only relevantto recruit DCs into uninflamed tissues (6). This may explainwhy CXCL17 was dispensable for chemotaxis of mDCs to thelung of M. tuberculosis HN878-infected CXCL17�/� mice,whereas a higher number of mDCs accumulated into the lungof mice receiving exogenous rCXCL17 since early dpi, whenthe degree of inflammation was low. Alternatively, our findings

FIGURE 3. M. tuberculosis HN878 infection in CXCL17�/� mice.

WT B6 and CXCL17�/� mice were aerosol-infected with �100 CFU of M. tuberculosis HN878 and sacrificed at given dpi (n 5 5 per group per time

point). (A) Lung myeloid cell populations were enumerated in M. tuberculosis HN878-infected WT and CXCL17�/� mice using flow cytometry at 30

dpi. (B) Lung bacterial burden was determined by plating and counting CFUs at 30, 100, and 150 dpi. (C) Lung area occupied by inflammation was

quantified in H&E-stained, formalin-fixed, and paraffin-embedded (FFPE) lungs using NanoZoomer software. The percentage of lung area occupied

by inflammation per mouse lung was determined at 100 dpi. (D) Representative microphotographs showing H&E-stained FFPE lung sections from

M. tuberculosis HN878-infected WT and CXCL17�/� mice at 100 dpi. Comparisons between groups were performed using the unpaired Student t

test at each time point. The data shown represent mean (6 SD) values from two to three independent experiments. *p # 0.05, **p # 0.01.

FIGURE 4. Bactericidal activity of CXCL17 against M. tuberculosis.

Exponentially growing M. tuberculosis HN878 (A) and M. tuberculosis H37Rv (B) were treated with human rCXCL17 (see Materials and Methods).

After chemokine exposure, the bacteria were spread on 7H11 agar solid medium plates and incubated at 37�C for 14–21 d. Surviving bacteria were

counted as CFU/ml at each concentration. Saline solution and rifampicin (Rif) were used as negative and positive controls, respectively. Data were

analyzed using the Kruskal–Wallis test and the post hoc Dunn multiple comparisons test. *p # 0.05, ***p # 0.001.

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suggest that, besides CXCL17, DCs may follow other redundantchemotactic signals to accumulate at the sites of M. tuberculosisexposure in mice (1).

Our data also show that a proportion of monocytes use theCXCL17 axis to infiltrate the lung during TB. Although theidentity of the CXCL17R is yet controversial, monocytesexpressing the G protein�coupled receptor 3 (GPR35), the pro-posed CXCL17R (16), might represent the myeloid populationthat migrated to the M. tuberculosis�infected lung in ourexperiments. GPR35 is constitutively expressed in inflammatorymacrophages and downregulated upon LPS stimulation (17).Unfortunately, we did not address the expression of this recep-tor on monocytes/macrophages. Furthermore, little literatureexists about a role for GPR35 during TB. However, our resultsalso indicate that the subsets of monocytes responding toCXCL17 and their macrophage progeny may not exert relevantfunctions against M. tuberculosis, as their absence did notincrease lung bacterial burden in CXCL17�/� mice.

Other monocyte and macrophage subsets recruited by dis-tinct chemokines may be more important for protective immu-nity against TB and compensate for the lack of CXCL17 activityin mice. Indeed, different myeloid cell subpopulations display avariable capacity to restrict M. tuberculosis growth (18). Thisfunctional variability depends on the origin of the phagocytes(18) and, perhaps, also on the chemokines mediating theirrecruitment, although the same chemokine axis can mediatethe recruitment of permissive and resistant myeloid cell sub-sets. For instance, CCR21 AMs mediate protective immunityagainst M. tuberculosis (7), whereas monocyte-derived macro-phages responding to CCL-2 are more permissive and contrib-ute to disease pathogenesis (19, 20). Collectively, these datasuggest that the chemotactic capacity of CXCL17 is dispensablefor control of M. tuberculosis HN878 infection in mice.

In contrast, our human data suggest a possible protectiverole for CXCL17 that is lost during PTB. This discrepancymight be related to a possible capacity of mice to make a com-pensatory response in the absence of CXCL17 through the pro-duction of other chemokines or cytokines that regulatemigration or bactericidal activity of myeloid cells, as mentionedabove. It is also probable that the levels of CXCL17 are downre-gulated in humans during PTB because of the increased activa-tion of other chemokine axes. Conversely, some activities ofCXCL17, such as its chemotactic functions, may be moreimportant in humans than in mice. Thus, future studies shouldevaluate the capacity of myeloid cells from patients with differ-ent forms of TB to migrate in response to CXCL17.

Bactericidal activity of CXCL17 against respiratory microor-ganisms (3), including M. tuberculosis, might also differ betweenhumans and mice. Interestingly, high expression of CXCL17 isobserved at the luminal surface of the human alveolar epithe-lium, where it could act as an antimicrobial peptide (2, 3). Toour knowledge, the bactericidal capacity of CXCL17 to lyse M.tuberculosis has not been tested before. Nonetheless, our resultsdemonstrate that human CXCL17 lacks a bactericidal capacityto directly kill M. tuberculosis in vitro. Moreover, our findings

in CXCL17�/� mice also indicate that this activity is not rele-vant for M. tuberculosis clearance in vivo. Despite this, furtheranalyses are required to evaluate whether CXCL17 potentiatesthe bactericidal activity of human and mouse macrophages orchanges their activation status when challenged with M. tuber-culosis. Importantly, the role of CXCL17 in TB immunity shouldbe further addressed in murine models of infection with differ-ent M. tuberculosis strains and in larger cohorts of TB patientsas well.

In summary, our data revealed that M. tuberculosis infectioninduces the production of CXCL17 in the mouse lungs in vivoand by epithelial and myeloid cells in vitro. In addition,CXCL17 recruits monocytes to sites of M. tuberculosis infectioninside mouse lungs. In humans, lower serum CXCL17 levels areobserved among active PTB patients when compared with sub-jects with LTBI, suggesting a protective role. However, theadministration of exogenous CXCL17 to WT mice and theabsence of CXCL17 signaling in knockout animals do not alterthe susceptibility to TB in the murine model of hypervirulentM. tuberculosis HN878 infection. These findings suggest that,despite robust induction of CXCL17, this chemokine may havea redundant role during M. tuberculosis infection. Hence, ourstudy expands the current knowledge of chemokine functionsduring TB.

DISCLOSURES

The current study was conducted in the absence of any com-mercial or financial relationships that could be construed as apotential conflict of interest. The authors have no financial con-flicts of interest.

ACKNOWLEDGMENTS

We thank Dr. Shyamala Thirunavukkarasu and Dr. Shibali Das from theDepartment of Molecular Microbiology, Washington University Schoolof Medicine, St. Louis, for scientific critique of the manuscript.

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