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Article

Dectin-1-Independent Macrophage Phagocytosis of Mycobacterium abscessus

by
Alma E. Ochoa
,
Jack H. Congel
,
Jodi M. Corley
,
William J. Janssen
,
Jerry A. Nick
,
Kenneth C. Malcolm
and
Katherine B. Hisert
*
Department of Medicine, National Jewish Health, 1400 Jackson Street, Room A550, Denver, CO 80206, USA
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2023, 24(13), 11062; https://doi.org/10.3390/ijms241311062
Submission received: 17 May 2023 / Revised: 25 June 2023 / Accepted: 29 June 2023 / Published: 4 July 2023
(This article belongs to the Special Issue Macrophages in Respiratory Diseases)
Browse Figures
Versions Notes

Abstract

:
Mycobacterium abscessus, a species of nontuberculous mycobacteria (NTM), is an opportunistic pathogen that is readily cleared by healthy lungs but can cause pulmonary infections in people with chronic airway diseases. Although knowledge pertaining to molecular mechanisms of host defense against NTM is increasing, macrophage receptors that recognize M. abscessus remain poorly defined. Dectin-1, a C-type lectin receptor identified as a fungal receptor, has been shown to be a pathogen recognition receptor (PRR) for both M. tuberculosis and NTM. To better understand the role of Dectin-1 in host defense against M. abscessus, we tested whether blocking Dectin-1 impaired the uptake of M. abscessus by human macrophages, and we compared M. abscessus pulmonary infection in Dectin-1-deficient and wild-type mice. Blocking antibody for Dectin-1 did not reduce macrophage phagocytosis of M. abscessus, but did reduce the ingestion of the fungal antigen zymosan. Laminarin, a glucan that blocks Dectin-1 and other PRRs, caused decreased phagocytosis of both M. abscessus and zymosan. Dectin-1−/− mice exhibited no defects in the control of M. abscessus infection, and no differences were detected in immune cell populations between wild type and Dectin-1−/− mice. These data demonstrate that murine defense against M. abscessus pulmonary infection, as well as ingestion of M. abscessus by human macrophages, can occur independent of Dectin-1. Thus, additional PRR(s) recognized by laminarin participate in macrophage phagocytosis of M. abscessus.

1. Introduction

Nontuberculous mycobacteria (NTM) are considered opportunistic pathogens because they are ubiquitous in the environment [1] and do not cause disease in healthy individuals. However, NTM can cause infections in people with chronic airway diseases, and those with bronchiectasis have an up to 10,000-fold greater risk of infection compared to the general population [2]. Between 10 and 37% of people with chronic airways disease are estimated to have NTM chronically present in their airways, with Mycobacterium avium and Mycobacterium abscessus being the two predominant NTM species identified [3,4,5]. The basis for increased susceptibility to pulmonary NTM infections in people with airways diseases is likely multifactorial but remains poorly understood. This knowledge gap has become critical for several reasons. First, the prevalence of pulmonary NTM infections is increasing globally (a 2–6-fold increase between 1980s and 2010s) [3,6], with a marked increase in NTM in people with chronic airway diseases [4,5,7]. Second, pulmonary NTM infections worsen symptoms and outcomes in people with pre-existing lung diseases, including more rapid lung function decline and increased mortality [7,8]. Finally, pulmonary NTM infections, particularly those caused by M. abscessus, are difficult to treat, requiring administration of multiple antibiotics for months, with up to a 50% failure rate of eradication [4,9,10]. Accordingly, it is important to understand the mechanisms by which immune cells recognize and clear NTM from the lung.
Resistance to pulmonary NTM infections in the healthy lung relies on effective mucociliary clearance and intact innate immune responses, as defects in either increase susceptibility to these infections [11,12]. However, the ways in which immune cells in healthy lungs eliminate inhaled NTM remain a subject requiring continued study. Defense against mycobacteria is thought to be dependent on macrophages [13,14]. Considerable data exist regarding molecular mechanisms required to combat Mycobacterium tuberculosis infection [15,16,17]. It is unclear, though, if these findings can be extrapolated to NTM, as M. tuberculosis is an obligate intracellular pathogen, while NTM are opportunistic bacteria that can live outside the mammalian host. Several members of the C-type lectin receptor (CLR) family have been identified as important pattern recognition receptors (PRRs) used by macrophages to identify and mount inflammatory responses to mycobacteria; however, individual macrophage CLRs do not recognize all species of mycobacteria [17,18,19]. For example, Dectin-2 recognizes mannose-capped lipoarabinomannan (Man-LAM), a major lipoglycan found on the surface of M. tuberculosis and M. intracellulare, but does not recognize M. abscessus or M. smegmatis, neither of which employ mannose capping of lipoglycans [19]. Dectin-1, a CLR originally identified as an essential PRR for fungal antigens [20,21], has been shown to recognize several mycobacterial species including M. tuberculosis, M. bovis, M. avium, and M. abscessus [22,23,24], triggering macrophage intracellular signaling cascades and subsequent production of inflammatory cytokines [22,23,24]. One frequently cited study concluded that Dectin-1 is also essential for macrophage phagocytosis of M. abscessus [23]; however, macrophage PRRs that detect M. abscessus are overall less well studied than PRRs for M. tuberculosis. We sought to further characterize the role of Dectin-1 in host defense against initial infection with M. abscessus by evaluating the requirement for Dectin-1 during murine acute M. abscessus lung infection and human macrophage phagocytosis of M. abscessus.

2. Results

2.1. Blocking Antibody to Dectin-1 Reduces Phagocytosis of Zymosan by Human Monocyte Derived Macrophages but Does Not Decrease Uptake of M. abscessus

In people with chronic airways diseases and bronchiectasis, macrophages that accumulate in inflamed airways at sites of disease are primarily monocyte-derived macrophages (MDMs) recruited from the blood, rather than resident airspace or alveolar macrophages [25,26,27]. MDMs are therefore the macrophage population most likely to first interact with inhaled NTM. We thus chose to study the role of Dectin-1 on blood-derived human MDMs. Monocytes were isolated from healthy donors and matured to macrophages over seven days using macrophage-colony stimulating factor (M-CSF) as described [28]. Flow cytometry confirmed the expression of Dectin-1 on MDMs (Figure 1). To determine whether blocking Dectin-1 reduces phagocytosis of M. abscessus, we exposed MDMs to either a Dectin-1-specific blocking antibody (clone 259931) or isotype control antibody, infected cells with M. abscessus for 30 min, and then lysed the cells to determine internalized colony forming units (CFUs). As shown in Figure 2A, although there was variability in the uptake of M. abscessus by macrophages from different donors, there was no significant change in internalized M. abscessus when MDMs were pre-treated with Dectin-1 blocking antibody compared to the isotype control antibody.
To validate the ability of this specific antibody clone (259931) to block Dectin-1 [29,30], we evaluated whether it could reduce phagocytosis of zymosan, a β-glucan-rich cell wall component from the yeast Saccharomyces cerevisiae that requires Dectin-1 for ingestion [31]. We treated MDMs with either isotype control or clone 259931, and then incubated cells with green fluorescent protein (GFP)-expressing M. abscessus or fluorescently labeled (AF488) zymosan. Pre-treatment of MDMs with clone 259931 significantly reduced the uptake of AF488 zymosan by MDMs, but did not decrease the ingestion of GFP-M. abscessus (Figure 2B,D, Table 1). Furthermore, flow cytometry demonstrated a significant reduction in exposed Dectin-1 on the surface of MDMs treated with clone 259931 (with or without incubation with fluorescent microbial particles) compared to MDMs treated with the isotype control (Figure 2C, Table 1). These data demonstrate that the anti-Dectin-1 antibody clone 259931 blocks Dectin-1, obscuring it from subsequent antibodies that detect Dectin-1 and preventing Dectin-1 from binding the β-glucan-rich particle zymosan. Furthermore, human MDMs experience no impairment in the phagocytosis of M. abscessus following the specific blockade of Dectin-1.

2.2. Laminarin Decreases Phagocytosis of Both M. abscessus and Zymosan by Human Monocyte Derived Macrophages

In prior studies investigating the role of Dectin-1 in macrophage recognition of NTM, many authors have used the molecule laminarin to block Dectin-1 [22,23,24,32]. Laminarin is a low molecular weight β-glucan found in marine algae [33] which blocks Dectin-1 but has been shown to bind and block other PRRs and scavenger receptors as well [34,35]. Laminarin was found to decrease macrophage ingestion of M. abscessus [23]. Since we observed that specific blockade of Dectin-1 did not reduce phagocytosis of M. abscessus by MDMs, and prior studies investigating phagocytosis of M. abscessus used murine macrophages, we sought to determine whether laminarin decreases the ingestion of M. abscessus by human MDMs. We treated MDMs with laminarin or vehicle control (DMSO), and then incubated cells with GFP-expressing M. abscessus or AF488 zymosan. Laminarin reduced MDM phagocytosis of both zymosan particles and M. abscessus (Figure 3A,C, Table 2). As with the anti-Dectin-1 antibody clone 259931, the treatment of MDMs with laminarin decreased detectable Dectin-1 on the surface of MDMs (Figure 3B), confirming that laminarin blocks Dectin-1. These data, consistent with previously reported data from other researchers, suggest that a receptor (or receptors) that binds to and can be blocked by laminarin is required for macrophage phagocytosis of M. abscessus.

2.3. Dectin-1−/− Mice Do Not Exhibit Increased Susceptibility to M. abscessus Lung Infection

Our in vitro results using human macrophages demonstrate that Dectin-1 is not essential for the uptake of M. abscessus; however, work by others has shown that Dectin-1, in combination with TLR2, is required for macrophages to produce inflammatory cytokines in response to M. abscessus [22,23,24]. To determine whether Dectin-1 plays an essential role in host resistance to M. abscessus pulmonary infection, we infected wild-type and Dectin-1−/− mice with M. abscessus via the intratracheal route. Wild-type (WT) mice infected with between 106 and 107 colony forming units (CFUs) of M. abscessus cleared 1–2 logs of bacteria from the lungs during the first week of infection, with minimal CFUs remaining in the bronchoalveolar lavage (BAL) fluid at this time point (Figure 4A). Dectin-1−/− mice exhibited no differences in lung or BAL CFUs at one week after infection (Figure 4A). There were no differences detected between wild-type and Dectin-1−/− mice in total BAL cell numbers, or in relative or absolute abundance of BAL cells subsets as determined by microscopy (Figure 4B). Furthermore, staining of lung homogenates and BAL cells for flow cytometry revealed that the lack of Dectin-1 caused no alterations in proportions of immune cell populations in the lungs and in the airspaces following infection (Figure 4C,D, Table 3). The only difference detected between the WT and Dectin-1−/− mice was that Dectin-1 protein was not detectable on the surface of lung cells in the Dectin-1−/− mice (Figure 4E,F). These data indicate that Dectin-1 is not required for clearance of M. abscessus in a murine pulmonary infection model, and the absence of Dectin-1 does not appear to alter immune responses to M. abscessus.

3. Discussion

Inspired by prior studies describing Dectin-1 as an important PRR for M. abscessus [23,24], we evaluated the importance of Dectin-1 in recognition and phagocytosis of M. abscessus by macrophages and during a murine model of acute M. abscessus pulmonary infection. We found that human macrophages can ingest M. abscessus by methods independent of Dectin-1, and mice lacking Dectin-1 demonstrated no impairment in eradication of M. abscessus pulmonary infection. However, laminarin, a β-glucan that obstructs several PRRs in addition to Dectin-1, reduced macrophage phagocytosis of M. abscessus.
These data support two possible models of host recognition of M. abscessus. First, there may be redundancy in PRRs used by macrophages to phagocytose M. abscessus, and Dectin-1 is sufficient but not necessary to recognize and ingest M. abscessus. In support of this model, Shin et al. [23] found that over-expression of Dectin-1 in RAW264.7 cells resulted in enhanced uptake of M. abscessus compared to cells transfected with an empty vector. Alternately, a yet-to-be-identified receptor that is also blocked by laminarin may be the primary PRR used by macrophages to ingest M. abscessus, and this receptor may exhibit preferential binding to M. abscessus when multiple PRRs including Dectin-1 are present. Laminarin has been shown to block recognition of pathogens by receptors other than Dectin-1, including LYSMD3, CD11b, and ephrin-A2 (EphA2) [34,36,37,38], and thus one or more of these receptors may be the critical PRR for M. abscessus.
Dectin-1-deficient mice did not demonstrate increased susceptibility to acute pulmonary infection by M. abscessus, and no aberrant immune responses were detected in mice lacking Dectin-1. We acknowledge that our immune phenotyping did not include all immune pathways, and that Dectin-1−/− may manifest subtle differences in immune phenotypes after infection with M. abscessus. Furthermore, mice in which the CLEC7A gene (that encodes Dectin-1) has been disrupted in the germline, and have thus lacked Dectin-1 during development, may acquire compensatory upregulation of other receptors; however, if this compensation does occur, it would further support the theory that there is redundancy in PRRs that recognize M. abscessus, and that recognition can occur independent of Dectin-1. Our use of ex vivo receptor blocking experiments demonstrated that acute blockade of Dectin-1 did not impair macrophage phagocytosis, also consistent with Dectin-1-independent recognition mechanisms of M. abscessus.
Prior studies demonstrated that Dectin-1-deficient mice did not exhibit increased susceptibility to pulmonary M. tuberculosis infection [39]. Although several in vitro studies demonstrated that blocking Dectin-1 decreased macrophage and dendritic cell cytokine expression after exposure to M. tuberculosis [22,23,24], Dectin-1-deficient mice did not show increased bacterial burden in the lungs following aerosol infection with M. tuberculosis, and there were no observed changes in murine survival, histopathology of lung lesions, or inflammatory responses in M. tuberculosis-infected Dectin-1−/− mice compared to wild-type mice. In contrast, mice lacking the C-type lectin receptor CLEC4D (also known as MCL) were impaired in their control of bacterial replication with more CFUs in their lungs than WT mice after infection with either aerosolized H37Rv M. tuberculosis or M. bovis BCG, and pulmonary neutrophil infiltration increased [40]. Laminarin does not bind to or block CLEC4D, and thus CLEC4D is unlikely to be the PRR responsible for recognition and uptake of M. abscessus; however, these studies demonstrate the importance of extending in vitro host–pathogen interaction data to in vivo models of infection.
It should also be noted that cells may use distinct PPRs (or groups of PRRs), including Toll-like receptors (TLRs), C-type lectins, scavenger receptors, and others, to recognize different mycobacterial species [41,42,43,44]. As indicated above, although Dectin-1 was not essential for protection against murine infection with M. tuberculosis [17], CLEC4D/MCL was found to be important for murine defense against M. tuberculosis [40]. Dectin-2, which recognizes mannose-capped lipoarabinomannan (Man-LAM), participates in host recognition of M tuberculosis and M. intracellulare, but not M. abscessus or M. smegmatis, neither of which employ mannose capping of lipoglycans [19]. Although our data suggest that Dectin-1 may be dispensable for macrophage recognition of and host defense against M. abscessus, other work has shown a role for Dectin-1 in keratinocyte recognition and phagocytosis of M. ulcerans [32]. Finally, individual mycobacterial molecules may be recognized by several host PRRs or proteins: M. avium and some other species of NTM have been shown to produce fibronectin-binding proteins (FAPs) that interact with the host in multiple ways: adhering to extracellular matrix proteins [45], binding to integrins on host cells [46], and interacting directly with immune cell TLRs [47].
We acknowledge limitations to this study, which was focused on relatively acute events in host response to M. abscessus. It is possible that Dectin-1 could contribute to later stages of infection, or be relevant when different multiplicity of infection or greater inocula are used. Also, these studies were focused on the normal response to M. abscessus, relevant to the general population that is not susceptible to the infection. It is possible that in at-risk populations, Dectin-1-mediated responses to M. abscessus could be of greater (or less) clinical significance. We also note that we did not see complete inhibition of phagocytosis of M. abscessus by laminarin, or of zymosan by either laminarin or the anti-Dectin-1 antibody. The extent of blocking of PRRs with either laminarin or blocking antibodies is a stoichiometry challenge, and depends on both number of host cells and concentration of blocking reagent. Our studies showed a ~10% absolute reduction and a ~25% relative reduction in microbe or particle uptake when MDMs were incubated with laminarin at the concentrations we used. This is consistent with other studies in which laminarin was used to block the uptake of β-glucan particles and mycobacteria by different cell types (neutrophils, keratinocytes, and macrophages) [32,34,36].
In conclusion, our data demonstrate the existence of Dectin-1-independent pathways for macrophage recognition and phagocytosis of M. abscessus, an opportunistic pathogen with increasing global disease burden. These results highlight a fundamental gap in knowledge of how the healthy immune system eradicates M. abscessus, which is essential information for helping understand why specific populations, including those with chronic airways diseases, are at an increased risk for M. abscessus pulmonary infection.

4. Materials and Methods

4.1. Preparation of M. abscessus Inocula for Infections of Cells and Mice

M. abscessus ATCC 19977 smooth variant or M. abscessus ATCC 19977 smooth variant that expresses the green fluorescent protein (GFP) originally carried on the pBCM-2 vector were used to infect mice, and human-monocyte-derived macrophages (MDMs). Liquid aliquots of 7H9 Middlebrook media (Sigma-Aldrich, St. Louis, MI, USA, catalog number: M0178) supplemented with 10% of the Middlebrook ADC (albumin/dextrose/catalase) growth supplement (Millipore Sigma, Burlington, MA, USA, catalog number: M0553) were inoculated with M. abscessus or GFP-M. abscessus. Liquid cultures were incubated at 37 °C, shaking at 225 RPM over 5 days.
For infection of monocyte-derived macrophages (MDMs), stationary phase cultures (5-day liquid cultures) of M. abscessus or GFP-M. abscessus were sonicated 3–5 times for 1 s using a power of 3–5 (Fisher Scientific (Hampton, NH, USA) Sonic Dismembrator model 100). For inoculation of mice, M. abscessus stationary phase cultures were sub-cultured 1:5 in Middlebrook 7H9 broth base media supplemented with 10% Middlebrook ADC growth supplement, and grown for 17–20 h (on the day prior to infection). On the day of infection, sub-cultures were sonicated 3–5 times for 1 s using a power of 3–5. Following sonication, bacterial inocula for either MDMs or mice were pelleted 8000× g for 5 min, cells were washed 3 times with 1 mL of 1× PBS and resuspended in 500 μL of 1× PBS. Optical density was determined using a Beckman (Brea, CA, USA) DU 640 Spectrophotometer with absorbance set to 600 nm. M. abscessus cultures prepared as described above were diluted with a 1× PBS until an optical density (OD) of 1.0 was achieved. Prior experiments determined that an OD600 of 1.0 is equal to 2–4 × 108 CFU/mL. Cultures were then diluted to achieve a final goal inoculum of a ~5 × 106 CFU in 50 mL.

4.2. Preparation of Zymosan Inoculum

To prepare zymosan A (S. cerevisiae) BioParticles, Alexa Fluor 488 conjugate (Thermo Fisher (Waltham, MA, USA) catalog number: Z23373) lyophilized particles were reconstituted in 1 mL of 1× PBS, vigorously vortexed, and passed through a 27 G needle 5 times to disrupt clumps. Zymosan was then diluted in an RPMI 1640 medium + Glutamax supplemented with a 10% fetal bovine serum (FBS) and sonicated 3 times using a power of 3–5.

4.3. Isolation of Human Monocyte Derived Macrophages (MDMs)

Blood was obtained with IRB approval from healthy volunteers. PBMC were isolated from blood using density gradient centrifugation with Ficoll-Paque Plus (Cytiva; Marlborough, MA, USA, catalog number: 17144002). PBMCs were washed 3 times using cold 1× PBS with a 2.5 mM EDTA. Monocytes were isolated from PBMCs via negative selection using the pan monocyte isolation kit (Miltenyi Biotec; Gaithersburg, MD, USA, catalog number: 130-096-537) according to the manufacturer’s instructions using the manual isolation protocol. MDMs were plated at a density of 1.25 × 105 cells/well in a 96-well plate or 2.5 × 105 cells/well in a 48-well plate (for the quantitation of phagocytosis using bacterial CFUs) or 5 × 105 cells in a 24-well plate for the quantitation of phagocytosis using flow cytometry. For confocal microscopy, 3 × 105 monocytes were plated on glass coverslips. Monocytes were matured to MDMs as described [28]. Briefly, cells were cultured in an RPMI 1640 medium + Glutamax supplement (Fisher catalog number: 61-870-036) with 1% penicillin/streptomycin (Sigma catalog number: P4333) + 10% fetal bovine serum (FBS), and 100 ng/mL of recombinant human M-CSF (Peprotech; Rocky Hill, NJ, USA, catalog number: 30025) for 7 days. Media were replaced every two days.

4.4. Monocyte-Derived Macrophage Pathogen Recognition Receptor Blocking Assay

MDMs were washed 3× to remove residual penicillin/streptomycin, and then incubated for 1 h at 37 °C in a CO2 incubator with 5 μg/mL or 20 μg/mL of the Human anti-Dectin-1/CLEC7A Antibody (R&D Systems (R&D Systems (Minneapolis, MN, USA) catalog number: MAB1859, antibody clone #259931) or the Mouse IgG2B Isotype Control (R&D Systems catalog number: MAB004). Alternately, cells were incubated in laminarin 0.5–2 mg/mL (Thermo Fisher catalog number: J66193.MD) or 0.5–2% of DMSO as vehicle control for 1 h at 37 °C in a CO2 incubator. Cells were then washed, and media were added to MDMs containing either M. abscessus (MOI 10:1) or zymosan particles with a final inoculation ratio of ~0.5 (1.5 × 105 particles added to 2.5 × 105 MDMs). MDMs were centrifuged at 1000× g for 1 min to enhance microbial contact, then incubated for 30 min (or 60 min for MDMs on glass coverslips) at 37 °C in a CO2 incubator. After 30 min, media were changed on MDMs infected with M. abscessus to media containing amikacin 250 μg/mL to kill extracellular bacteria. For the uptake determined by intracellular CFUs, MDMs were washed 3 times with 1× PBS and lysed with 0.1% triton. Each sample was sonicated 3 times for 1 s (power of 3–5) before serial dilution and plating on LB agar. For flow cytometry, cells were washed with room temperature 1× PBS, and then detached by incubating in 4 °C 2.5 mM EDTA for 5 min followed by using a cell scraper, fixed blade (Cytoone, Ocala, FL, USA, catalog number: CC7600-0220).

4.5. Flow Cytometry of Monocyte-Derived Macrophages

Following detachment of MDMs from tissue culture plates, cells were incubated with FcR blocking reagent (Miltenyi Biotec catalog number: 130-059-901), and then stained using a mix of fluorochrome conjugated antibodies listed in Table 4. Cells were fixed with 4% PFA (Electron Microscopy Sciences; Hatfield, PA, USA, catalog number: 15710-S). Samples were acquired on an LSRII flow cytometer. Flow cytometry data were analyzed using the FlowJo v.10.7.1 Software. Gating strategy is shown in Supplementary Figure S1.

4.6. Staining of Monocyte-Derived Macrophages for Confocal Microscopy

After 1 h of incubation with GFP-M. abscessus or AF88-Zymosan, MDMs were washed 3 times. Lycopersicon Esculentum (Tomato) Lectin (LEL, TL), DyLight 594 (Thermofisher catalog number: L32471) was used to stain cell membranes. Cells were fixed with 4% PFA (Electron Microscopy Sciences catalog number: 15710-S). Coverslips were mounted Vectashield Antifade Mounting Medium with DAPI (Vector Laboratories (Newark, CA, USA) catalog number: H-1800). Slides were imaged using a Zeiss LSM 700 (Zeiss, Jena, Germany) confocal microscope.

4.7. Murine Model of M. abscessus Pulmonary Infection

All mice listed were approved by the Institutional Animal Care and Use Committee (IACUC) (Protocol number: AS2574-02-23) at National Jewish Health. The B6.129S6-Clec7atm1Gdb/J (Dectin-1 KO) and C57BL/6J (WT) mice strains were purchased from Jackson Labs. Male mice, 8–9 weeks of age, were inoculated with 50 μL containing 1 × 106 to 5 × 106 CFUs of M. abscessus via oropharyngeal aspiration. The murine pulmonary infection model was performed two times, with 5 mice per group in each experiment. Oropharyngeal inoculation was performed as described [48]. Briefly, mice were anesthetized mice with Fluriso isoflurane (VET one; Boise, ID, USA, catalog number: 501017) and positioned vertically. The tongue was gently pulled out of the mouth laterally to cover the esophagus and minimize obstruction of the trachea. M. abscessus in 50 μL PBS was deposited in the oropharynx; this inoculum was aspirated into the lungs during the mouse’s subsequent inhalation. On day 6 after infection, mice were euthanized via intraperitoneal injection of barbiturate solution (Fatal-Plus). The left lung main stem bronchus was tied off, and the left lung was removed for homogenization and plating for CFUs. Bronchoalveolar lavage (BAL) was performed on the right lung to plate for CFU, calculate total cell counts and differentials, and to use for flow cytometry. Following BAL, the right lung homogenate was perfused with saline to eliminate blood, recovered, and digested to make a single-cell suspension for flow cytometry.

4.8. Lung Tissue Homogenization to Determine Bacterial Burden

Lungs were bead beaten with 0.1% triton with stainless steel beads (Next Advance catalog number: SSB14B) using the bullet blender (Next Advance (Troy, NY, USA) catalog number: BT12LT) twice for 3 min at level 7–8. Homogenized lungs were serially diluted with 1× PBS and plated for CFUs on LB agar media plates (BD (Franklin Lakes, NJ, USA) catalog number: 244510), supplemented with 2.5 μg/mL of amphotericin B antifungal (Corning (Corning, NY, USA) catalog number: 30-003-CF).

4.9. Flow Cytometry on Murine Bronchoalveolar Lavage and Lung Tissue Homogenate Cells

The right lungs were minced into small pieces using scissors and incubated with 20% of liberase (Millipore Sigma catalog number: 05401127001) in HBSS for 25 min at 37 °C. All samples were kept on ice after this incubation. Lung digests were pressed through 70 μm cells to create single-cell suspensions. Cells were pelleted, resuspended, and cell numbers were quantitated. BAL and lung homogenate cells were resuspended with FcR block anti-mouse CD6/32 clone: 93 (Biolegend catalog number: 101320). Cells were then stained using a mix of fluorochrome conjugated antibodies shown in Table 5. Cells were washed and fixed with 4% PFA. Samples were acquired on an LSRII flow cytometer. Flow cytometry data were analyzed using the FlowJo v.10.7.1 Software (Ashland, OR, USA). Gating strategy is shown in Supplementary Data, Figures S2 and S3.

Supplementary Materials

The supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms241311062/s1.

Author Contributions

Conceptualization, W.J.J., J.A.N., K.C.M. and K.B.H.; Data curation, A.E.O., J.H.C. and K.B.H.; Formal analysis, A.E.O., J.H.C. and K.B.H.; Funding acquisition, K.B.H.; Investigation, A.E.O., J.H.C., J.M.C. and K.B.H.; Methodology, A.E.O., J.H.C., J.M.C. and K.B.H.; Project administration, K.B.H.; Resources, J.M.C., W.J.J., J.A.N., K.C.M. and K.B.H.; Supervision, K.B.H.; Validation, A.E.O. and K.B.H.; Writing—original draft, K.B.H.; Writing—review & editing, A.E.O., J.H.C., J.M.C., W.J.J., J.A.N. and K.C.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Cystic Fibrosis Foundation, grant number HISERT19R3, and the NIH, grant number K08 HL136786-01A1.

Institutional Review Board Statement

The study was conducted in accordance with the Declaration of Helsinki, and approved by the Biomedical Research Alliance of New York Institutional Review Board (HS1285-528). The animal study protocol was approved by the Institutional Animal Care and Use Committee (IACUC) (Protocol number: AS2574-02-23) at National Jewish Health.

Informed Consent Statement

We comply with the Declaration of Helsinki and Good Clinical Practice guidelines. Subjects are 21 years or older with no history of chronic diseases, or conditions that require routine, or daily medications. All subjects were given a written consent and blood was drawn into EDTA vacutainer tubes, 13 mM sodium citrate and no additive tubes.

Data Availability Statement

The data presented in this study are available on request from the corresponding author. Please send data requests to: Katherine Hisert, Department of Medicine, National Jewish Health; [email protected].

Acknowledgments

The authors thank Krystin Marlin and Ari Simenauer for assistance with preliminary experiments; Josh Loomis, from the flow cytometry and microscopy core at National Jewish Health; and Emily Wheeler for help with blood sample acquisition.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Honda, J.R.; Virdi, R.; Chan, E.D. Global Environmental Nontuberculous Mycobacteria and Their Contemporaneous Man-Made and Natural Niches. Front. Microbiol. 2018, 9, 2029. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  2. Honda, J.R.; Alper, S.; Bai, X.; Chan, E.D. Acquired and genetic host susceptibility factors and microbial pathogenic factors that predispose to nontuberculous mycobacterial infections. Curr. Opin. Immunol. 2018, 54, 66–73. [Google Scholar] [CrossRef] [PubMed]
  3. Adjemian, J.; Daniel-Wayman, S.; Ricotta, E.; Prevots, D.R. Epidemiology of Nontuberculous Mycobacteriosis. Semin. Respir. Crit. Care Med. 2018, 39, 325–335. [Google Scholar] [CrossRef] [PubMed]
  4. Martiniano, S.L.; Nick, J.A.; Daley, C.L. Nontuberculous Mycobacterial Infections in Cystic Fibrosis. Thorac. Surg. Clin. 2019, 29, 95–108. [Google Scholar] [CrossRef] [PubMed]
  5. Mirsaeidi, M.; Hadid, W.; Ericsoussi, B.; Rodgers, D.; Sadikot, R.T. Non-tuberculous mycobacterial disease is common in patients with non-cystic fibrosis bronchiectasis. Int. J. Infect. Dis. 2013, 17, e1000–e1004. [Google Scholar] [CrossRef] [Green Version]
  6. Ratnatunga, C.N.; Lutzky, V.P.; Kupz, A.; Doolan, D.L.; Reid, D.W.; Field, M.; Bell, S.C.; Thomson, R.M.; Miles, J.J. The Rise of Non-Tuberculosis Mycobacterial Lung Disease. Front. Immunol. 2020, 11, 303. [Google Scholar] [CrossRef] [Green Version]
  7. Pyarali, F.F.; Schweitzer, M.; Bagley, V.; Salamo, O.; Guerrero, A.; Sharifi, A.; Campos, M.; Quartin, A.; Mirsaeidi, M. Increasing Non-tuberculous Mycobacteria Infections in Veterans With COPD and Association With Increased Risk of Mortality. Front. Med. 2018, 5, 311. [Google Scholar] [CrossRef]
  8. Esther, C.R., Jr.; Esserman, D.A.; Gilligan, P.; Kerr, A.; Noone, P.G. Chronic Mycobacterium abscessus infection and lung function decline in cystic fibrosis. J. Cyst. Fibros. 2010, 9, 117–123. [Google Scholar] [CrossRef] [Green Version]
  9. Henkle, E.; Novosad, S.A.; Shafer, S.; Hedberg, K.; Siegel, S.A.R.; Ku, J.; Varley, C.; Prevots, D.R.; Marras, T.K.; Winthrop, K.L. Long-Term Outcomes in a Population-based Cohort with Respiratory Nontuberculous Mycobacteria Isolation. Ann. Am. Thorac. Soc. 2017, 14, 1120–1128. [Google Scholar] [CrossRef]
  10. Park, I.K.; Olivier, K.N. Nontuberculous mycobacteria in cystic fibrosis and non-cystic fibrosis bronchiectasis. Semin. Respir. Crit. Care Med. 2015, 36, 217–224. [Google Scholar] [CrossRef]
  11. Bustamante, J.; Boisson-Dupuis, S.; Abel, L.; Casanova, J.L. Mendelian susceptibility to mycobacterial disease: Genetic, immunological, and clinical features of inborn errors of IFN-gamma immunity. Semin. Immunol. 2014, 26, 454–470. [Google Scholar] [CrossRef] [Green Version]
  12. Fowler, C.J.; Olivier, K.N.; Leung, J.M.; Smith, C.C.; Huth, A.G.; Root, H.; Kuhns, D.B.; Logun, C.; Zelazny, A.; Frein, C.A.; et al. Abnormal nasal nitric oxide production, ciliary beat frequency, and Toll-like receptor response in pulmonary nontuberculous mycobacterial disease epithelium. Am. J. Respir. Crit. Care Med. 2013, 187, 1374–1381. [Google Scholar] [CrossRef] [Green Version]
  13. Johansen, M.D.; Herrmann, J.L.; Kremer, L. Non-tuberculous mycobacteria and the rise of Mycobacterium abscessus. Nat Rev. Microbiol. 2020, 18, 392–407. [Google Scholar] [CrossRef]
  14. Prasla, Z.; Sutliff, R.L.; Sadikot, R.T. Macrophage Signaling Pathways in Pulmonary Nontuberculous Mycobacteria Infections. Am. J. Respir. Cell Mol. Biol. 2020, 63, 144–151. [Google Scholar] [CrossRef]
  15. Singhania, A.; Wilkinson, R.J.; Rodrigue, M.; Haldar, P.; O’Garra, A. The value of transcriptomics in advancing knowledge of the immune response and diagnosis in tuberculosis. Nat. Immunol. 2018, 19, 1159–1168. [Google Scholar] [CrossRef]
  16. Tiberi, S.; du Plessis, N.; Walzl, G.; Vjecha, M.J.; Rao, M.; Ntoumi, F.; Mfinanga, S.; Kapata, N.; Mwaba, P.; McHugh, T.D.; et al. Tuberculosis: Progress and advances in development of new drugs, treatment regimens, and host-directed therapies. Lancet Infect. Dis. 2018, 18, e183–e198. [Google Scholar] [CrossRef]
  17. Marakalala, M.J.; Graham, L.M.; Brown, G.D. The role of Syk/CARD9-coupled C-type lectin receptors in immunity to Mycobacterium tuberculosis infections. Clin. Dev. Immunol. 2010, 2010, 567571. [Google Scholar] [CrossRef] [Green Version]
  18. Wagener, M.; Hoving, J.C.; Ndlovu, H.; Marakalala, M.J. Dectin-1-Syk-CARD9 Signaling Pathway in TB Immunity. Front. Immunol. 2018, 9, 225. [Google Scholar] [CrossRef]
  19. Yonekawa, A.; Saijo, S.; Hoshino, Y.; Miyake, Y.; Ishikawa, E.; Suzukawa, M.; Inoue, H.; Tanaka, M.; Yoneyama, M.; Oh-Hora, M.; et al. Dectin-2 is a direct receptor for mannose-capped lipoarabinomannan of mycobacteria. Immunity 2014, 41, 402–413. [Google Scholar] [CrossRef]
  20. Brown, G.D. Dectin-1: A signalling non-TLR pattern-recognition receptor. Nat. Rev. Immunol. 2006, 6, 33–43. [Google Scholar] [CrossRef]
  21. Taylor, P.R.; Tsoni, S.V.; Willment, J.A.; Dennehy, K.M.; Rosas, M.; Findon, H.; Haynes, K.; Steele, C.; Botto, M.; Gordon, S.; et al. Dectin-1 is required for beta-glucan recognition and control of fungal infection. Nat. Immunol. 2007, 8, 31–38. [Google Scholar] [CrossRef] [PubMed]
  22. Rothfuchs, A.G.; Bafica, A.; Feng, C.G.; Egen, J.G.; Williams, D.L.; Brown, G.D.; Sher, A. Dectin-1 interaction with Mycobacterium tuberculosis leads to enhanced IL-12p40 production by splenic dendritic cells. J. Immunol. 2007, 179, 3463–3471. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  23. Shin, D.M.; Yang, C.S.; Yuk, J.M.; Lee, J.Y.; Kim, K.H.; Shin, S.J.; Takahara, K.; Lee, S.J.; Jo, E.K. Mycobacterium abscessus activates the macrophage innate immune response via a physical and functional interaction between TLR2 and dectin-1. Cell Microbiol. 2008, 10, 1608–1621. [Google Scholar] [CrossRef] [PubMed]
  24. Yadav, M.; Schorey, J.S. The beta-glucan receptor dectin-1 functions together with TLR2 to mediate macrophage activation by mycobacteria. Blood 2006, 108, 3168–3175. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  25. Frankenberger, M.; Menzel, M.; Betz, R.; Kassner, G.; Weber, N.; Kohlhaufl, M.; Haussinger, K.; Ziegler-Heitbrock, L. Characterization of a population of small macrophages in induced sputum of patients with chronic obstructive pulmonary disease and healthy volunteers. Clin. Exp. Immunol. 2004, 138, 507–516. [Google Scholar] [CrossRef]
  26. Garratt, L.W.; Wright, A.K.; Ranganathan, S.C.; Grigg, J.; Sly, P.D.; behalf of AREST CF. Small macrophages are present in early childhood respiratory disease. J. Cyst. Fibros. 2012, 11, 201–208. [Google Scholar] [CrossRef] [Green Version]
  27. Schupp, J.C.; Khanal, S.; Gomez, J.L.; Sauler, M.; Adams, T.S.; Chupp, G.L.; Yan, X.; Poli, S.; Zhao, Y.; Montgomery, R.R.; et al. Single-Cell Transcriptional Archetypes of Airway Inflammation in Cystic Fibrosis. Am. J. Respir. Crit. Care Med. 2020, 202, 1419–1429. [Google Scholar] [CrossRef]
  28. Murray, P.J.; Allen, J.E.; Biswas, S.K.; Fisher, E.A.; Gilroy, D.W.; Goerdt, S.; Gordon, S.; Hamilton, J.A.; Ivashkiv, L.B.; Lawrence, T.; et al. Macrophage Activation and Polarization: Nomenclature and Experimental Guidelines. Immunity 2014, 41, 14–20. [Google Scholar] [CrossRef]
  29. Elder, M.J.; Webster, S.J.; Williams, D.L.; Gaston, J.S.; Goodall, J.C. TSLP production by dendritic cells is modulated by IL-1beta and components of the endoplasmic reticulum stress response. Eur. J. Immunol. 2016, 46, 455–463. [Google Scholar] [CrossRef]
  30. Li, D.; Dong, B.; Tong, Z.; Wang, Q.; Liu, W.; Wang, Y.; Liu, W.; Chen, J.; Xu, L.; Chen, L.; et al. MBL-mediated opsonophagocytosis of Candida albicans by human neutrophils is coupled with intracellular Dectin-1-triggered ROS production. PLoS ONE 2012, 7, e50589. [Google Scholar] [CrossRef]
  31. Brown, G.D.; Gordon, S. Immune recognition. A new receptor for beta-glucans. Nature 2001, 413, 36–37. [Google Scholar] [CrossRef]
  32. Lee, H.M.; Shin, D.M.; Choi, D.K.; Lee, Z.W.; Kim, K.H.; Yuk, J.M.; Kim, C.D.; Lee, J.H.; Jo, E.K. Innate immune responses to Mycobacterium ulcerans via toll-like receptors and dectin-1 in human keratinocytes. Cell Microbiol. 2009, 11, 678–692. [Google Scholar] [CrossRef]
  33. Karuppusamy, S.; Rajauria, G.; Fitzpatrick, S.; Lyons, H.; McMahon, H.; Curtin, J.; Tiwari, B.K.; O’Donnell, C. Biological Properties and Health-Promoting Functions of Laminarin: A Comprehensive Review of Preclinical and Clinical Studies. Mar. Drugs. 2022, 20, 772. [Google Scholar] [CrossRef]
  34. Bouchery, T.; Volpe, B.; Doolan, R.; Coakley, G.; Moyat, M.; Esser-von Bieren, J.; Wickramasinghe, L.C.; Hibbs, M.L.; Sotillo, J.; Camberis, M.; et al. beta-Glucan receptors on IL-4 activated macrophages are required for hookworm larvae recognition and trapping. Immunol. Cell Biol. 2022, 100, 223–234. [Google Scholar] [CrossRef]
  35. Jozefowski, S.; Yang, Z.; Marcinkiewicz, J.; Kobzik, L. Scavenger receptors and beta-glucan receptors participate in the recognition of yeasts by murine macrophages. Inflamm. Res. 2012, 61, 113–126. [Google Scholar] [CrossRef] [Green Version]
  36. van Bruggen, R.; Drewniak, A.; Jansen, M.; van Houdt, M.; Roos, D.; Chapel, H.; Verhoeven, A.J.; Kuijpers, T.W. Complement receptor 3, not Dectin-1, is the major receptor on human neutrophils for beta-glucan-bearing particles. Mol. Immunol. 2009, 47, 575–581. [Google Scholar] [CrossRef]
  37. He, X.; Howard, B.A.; Liu, Y.; Neumann, A.K.; Li, L.; Menon, N.; Roach, T.; Kale, S.D.; Samuels, D.C.; Li, H.; et al. LYSMD3: A mammalian pattern recognition receptor for chitin. Cell Rep. 2021, 36, 109392. [Google Scholar] [CrossRef]
  38. Yokoyama, C.C.; Baldridge, M.T.; Leung, D.W.; Zhao, G.; Desai, C.; Liu, T.C.; Diaz-Ochoa, V.E.; Huynh, J.P.; Kimmey, J.M.; Sennott, E.L.; et al. LysMD3 is a type II membrane protein without an in vivo role in the response to a range of pathogens. J. Biol. Chem. 2018, 293, 6022–6038. [Google Scholar] [CrossRef] [Green Version]
  39. Marakalala, M.J.; Guler, R.; Matika, L.; Murray, G.; Jacobs, M.; Brombacher, F.; Rothfuchs, A.G.; Sher, A.; Brown, G.D. The Syk/CARD9-coupled receptor Dectin-1 is not required for host resistance to Mycobacterium tuberculosis in mice. Microbes Infect. 2011, 13, 198–201. [Google Scholar] [CrossRef]
  40. Wilson, G.J.; Marakalala, M.J.; Hoving, J.C.; van Laarhoven, A.; Drummond, R.A.; Kerscher, B.; Keeton, R.; van de Vosse, E.; Ottenhoff, T.H.; Plantinga, T.S.; et al. The C-type lectin receptor CLECSF8/CLEC4D is a key component of anti-mycobacterial immunity. Cell Host. Microbe 2015, 17, 252–259. [Google Scholar] [CrossRef] [Green Version]
  41. Carvalho, N.B.; Oliveira, F.S.; Duraes, F.V.; de Almeida, L.A.; Florido, M.; Prata, L.O.; Caliari, M.V.; Appelberg, R.; Oliveira, S.C. Toll-like receptor 9 is required for full host resistance to Mycobacterium avium infection but plays no role in induction of Th1 responses. Infect. Immun. 2011, 79, 1638–1646. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  42. Killick, K.E.; Ni Cheallaigh, C.; O’Farrelly, C.; Hokamp, K.; MacHugh, D.E.; Harris, J. Receptor-mediated recognition of mycobacterial pathogens. Cell Microbiol. 2013, 15, 1484–1495. [Google Scholar] [CrossRef] [PubMed]
  43. Kim, W.S.; Yoon, J.H.; Shin, M.K.; Shin, S.J. Infection of Dendritic Cells with Mycobacterium avium subspecies hominissuis Exhibits a Functionally Tolerogenic Phenotype in Response to Toll-Like Receptor Agonists via IL-10/Cox2/PGE2/EP2 Axis. Front. Microbiol. 2019, 10, 1795. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  44. Marinho, F.A.; de Paula, R.R.; Mendes, A.C.; de Almeida, L.A.; Gomes, M.T.; Carvalho, N.B.; Oliveira, F.S.; Caliari, M.V.; Oliveira, S.C. Toll-like receptor 6 senses Mycobacterium avium and is required for efficient control of mycobacterial infection. Eur. J. Immunol. 2013, 43, 2373–2385. [Google Scholar] [CrossRef]
  45. Middleton, A.M.; Chadwick, M.V.; Nicholson, A.G.; Dewar, A.; Groger, R.K.; Brown, E.J.; Wilson, R. The role of Mycobacterium avium complex fibronectin attachment protein in adherence to the human respiratory mucosa. Mol. Microbiol. 2000, 38, 381–391. [Google Scholar] [CrossRef] [Green Version]
  46. Secott, T.E.; Lin, T.L.; Wu, C.C. Mycobacterium avium subsp. paratuberculosis fibronectin attachment protein facilitates M-cell targeting and invasion through a fibronectin bridge with host integrins. Infect. Immun. 2004, 72, 3724–3732. [Google Scholar] [CrossRef] [Green Version]
  47. Noh, K.T.; Shin, S.J.; Son, K.H.; Jung, I.D.; Kang, H.K.; Lee, S.J.; Lee, E.K.; Shin, Y.K.; You, J.C.; Park, Y.M. The Mycobacterium avium subsp. paratuberculosis fibronectin attachment protein, a toll-like receptor 4 agonist, enhances dendritic cell-based cancer vaccine potency. Exp. Mol. Med. 2012, 44, 340–349. [Google Scholar] [CrossRef] [Green Version]
  48. Altemeier, W.A.; Hung, C.F.; Matute-Bello, G. Mouse Models of Acute Lung Injury. In Acute Lung Injury and Repair; Schnapp, L.M., Feghali-Bostwick, C., Eds.; Respiratory Medicine; Springer International Publishing: Cham, Switzerland, 2017; pp. 5–23. [Google Scholar]
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Figure 1. Dectin-1 is expressed by human monocyte-derived macrophages (MDMs). Human MDMs were stained for flow cytometry. Dectin-1+ cells were identified by comparing viable CD14+ cells stained with anti-Dectin-1 antibody (blue) or a fluorescence minus one (FMO) panel lacking anti-Decitn-1 antibody (red). TLR2 and CD14 are shown to demonstrate that detection of only Dectin-1 is altered in the FMO panel.
Figure 1. Dectin-1 is expressed by human monocyte-derived macrophages (MDMs). Human MDMs were stained for flow cytometry. Dectin-1+ cells were identified by comparing viable CD14+ cells stained with anti-Dectin-1 antibody (blue) or a fluorescence minus one (FMO) panel lacking anti-Decitn-1 antibody (red). TLR2 and CD14 are shown to demonstrate that detection of only Dectin-1 is altered in the FMO panel.
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Figure 2. Blocking antibody for Dectin-1 does not impair MDM uptake of M. abscessus, but reduces phagocytosis of zymosan. MDMs were exposed to anti-Dectin-1 antibody (clone 259931) (α-D1) or isotype control (Iso) antibody, and then incubated with M. abscesuss or zymosan for 1 h. (A) Uptake of M. abscessus determined by quantification of intracellular colony forming units (CFUs). Each symbol represents a technical replicate. This experiment was repeated with MDMs from 4 different healthy donors, and representative results from 2 of the donors are shown. (B) Quantification of uptake of zymosan and M. abscessus determined by detection of fluorescent particles within macrophages by flow cytometry. Blue and green correspond to areas of lower cell density, red and orange are areas of high cell density, and yellow is mid-range. (C) Detection of Dectin-1 on the surface of MDMs by flow cytometry following treatment of MDMs with anti-Dectin-1 antibody (orange and red curve) or isotype control antibody (grey and blue curves). (D) Confocal fluorescent microscopy (63×) of representative images demonstrating decreased uptake of fluorescent zymosan particles in the presence of isotype control or anti-Dectin-1 blocking antibody. Blue = DAPI (stains DNA in nuclei); red = lectin (binds cell membranes); green = AF488 zymosan.
Figure 2. Blocking antibody for Dectin-1 does not impair MDM uptake of M. abscessus, but reduces phagocytosis of zymosan. MDMs were exposed to anti-Dectin-1 antibody (clone 259931) (α-D1) or isotype control (Iso) antibody, and then incubated with M. abscesuss or zymosan for 1 h. (A) Uptake of M. abscessus determined by quantification of intracellular colony forming units (CFUs). Each symbol represents a technical replicate. This experiment was repeated with MDMs from 4 different healthy donors, and representative results from 2 of the donors are shown. (B) Quantification of uptake of zymosan and M. abscessus determined by detection of fluorescent particles within macrophages by flow cytometry. Blue and green correspond to areas of lower cell density, red and orange are areas of high cell density, and yellow is mid-range. (C) Detection of Dectin-1 on the surface of MDMs by flow cytometry following treatment of MDMs with anti-Dectin-1 antibody (orange and red curve) or isotype control antibody (grey and blue curves). (D) Confocal fluorescent microscopy (63×) of representative images demonstrating decreased uptake of fluorescent zymosan particles in the presence of isotype control or anti-Dectin-1 blocking antibody. Blue = DAPI (stains DNA in nuclei); red = lectin (binds cell membranes); green = AF488 zymosan.
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Figure 3. Laminarin reduces MDM uptake of both M. abscessus and zymosan. MDMs were exposed to laminarin or vehicle (DMSO), and then incubated with M. abscesuss or zymosan for 1 h. (A) Quantification of uptake of zymosan and M. abscessus determined by detection of fluorescent particles within macrophages by flow cytometry. Blue and green correspond to areas of lower cell density, red and orange are areas of high cell density, and yellow is mid-range. (B) Detection of Dectin-1 on the surface of MDMs by flow cytometry following treatment of MDMs with laminarin (orange and red curve) or vehicle (grey and blue curves). (C) Confocal fluorescent microscopy (63×) of representative images demonstrating decreased uptake of fluorescent zymosan particles in the presence of laminarin. Blue = DAPI (stains DNA in nuclei); red = lectin (binds cell membranes); green = AF488 zymosan.
Figure 3. Laminarin reduces MDM uptake of both M. abscessus and zymosan. MDMs were exposed to laminarin or vehicle (DMSO), and then incubated with M. abscesuss or zymosan for 1 h. (A) Quantification of uptake of zymosan and M. abscessus determined by detection of fluorescent particles within macrophages by flow cytometry. Blue and green correspond to areas of lower cell density, red and orange are areas of high cell density, and yellow is mid-range. (B) Detection of Dectin-1 on the surface of MDMs by flow cytometry following treatment of MDMs with laminarin (orange and red curve) or vehicle (grey and blue curves). (C) Confocal fluorescent microscopy (63×) of representative images demonstrating decreased uptake of fluorescent zymosan particles in the presence of laminarin. Blue = DAPI (stains DNA in nuclei); red = lectin (binds cell membranes); green = AF488 zymosan.
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Figure 4. Dectin-1−/− mice do not exhibit increased susceptibility to pulmonary infection with M. abscessus. WT and Dectin-1−/− (D-KO) mice were infected via oropharyngeal inoculation with ~5 × 106 CFU M. abscessus. Mice were sacrificed at day 7 after infection. (A) M. abscessus colony forming units (CFUs) were measured from bronchoalveolar lavage (BAL) fluid and lung homogenates. Black bars represent average values. (B) BAL cell numbers and differentials were quantitated by microscopy. More variability was detected in host cell numbers than in bacterial CFUs, so results from 2 different experiments are shown in panel (B). Triangles represent individual animal values for WT mice; circles represent individual animal values for Dectin−/− mice. BAL cells and lung homogenates were stained for flow cytometry to measure relative abundance of immune cell sub-populations in the airspaces in the lung parenchyma. (C) Relative abundance of total leukocytes (CD45+ cells), neutrophils (CD45+Ly6G+), and total macrophages (CD45+Ly6G−CD88+CD64+) in BAL. Triangles represent individual animal values for WT mice; circle represent individual animal values for Dectin−/− mice (D) Relative abundance of resident (CD11c+SigLecF+) and recruited (CD11c−SigLecF−) macrophages in BAL Triangles represent individual animal values for WT mice; circles represent individual animal values for Dectin−/− mice (E) Relative expression of Dectin-1 on the surface of resident and recruited macrophages as measured by geometric mean fluorescence index of PerCP-Cy5.5-Dectin-1 antibody. Triangles represent individual animal values for WT mice; circles represent individual animal values for Dectin−/− mice; blue indicates recruited (recr) macrophages, and orange indicates resident (res) macrophages. (F) Representative histograms demonstrating lack of Dectin-1 expression on macrophages (CD45+Ly6G−CD88+CD64+ cells) and neutrophils (CD45+Ly6G+ cells) isolated from Dectin-1 KO mouse BAL.
Figure 4. Dectin-1−/− mice do not exhibit increased susceptibility to pulmonary infection with M. abscessus. WT and Dectin-1−/− (D-KO) mice were infected via oropharyngeal inoculation with ~5 × 106 CFU M. abscessus. Mice were sacrificed at day 7 after infection. (A) M. abscessus colony forming units (CFUs) were measured from bronchoalveolar lavage (BAL) fluid and lung homogenates. Black bars represent average values. (B) BAL cell numbers and differentials were quantitated by microscopy. More variability was detected in host cell numbers than in bacterial CFUs, so results from 2 different experiments are shown in panel (B). Triangles represent individual animal values for WT mice; circles represent individual animal values for Dectin−/− mice. BAL cells and lung homogenates were stained for flow cytometry to measure relative abundance of immune cell sub-populations in the airspaces in the lung parenchyma. (C) Relative abundance of total leukocytes (CD45+ cells), neutrophils (CD45+Ly6G+), and total macrophages (CD45+Ly6G−CD88+CD64+) in BAL. Triangles represent individual animal values for WT mice; circle represent individual animal values for Dectin−/− mice (D) Relative abundance of resident (CD11c+SigLecF+) and recruited (CD11c−SigLecF−) macrophages in BAL Triangles represent individual animal values for WT mice; circles represent individual animal values for Dectin−/− mice (E) Relative expression of Dectin-1 on the surface of resident and recruited macrophages as measured by geometric mean fluorescence index of PerCP-Cy5.5-Dectin-1 antibody. Triangles represent individual animal values for WT mice; circles represent individual animal values for Dectin−/− mice; blue indicates recruited (recr) macrophages, and orange indicates resident (res) macrophages. (F) Representative histograms demonstrating lack of Dectin-1 expression on macrophages (CD45+Ly6G−CD88+CD64+ cells) and neutrophils (CD45+Ly6G+ cells) isolated from Dectin-1 KO mouse BAL.
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Table
Table 1. Effects of Dectin-1 blocking antibody on MDM ingestion of zymosan and M. abscessus and on levels of exposed Dectin-1 protein. Table shows mean values with (standard errors).
Table 1. Effects of Dectin-1 blocking antibody on MDM ingestion of zymosan and M. abscessus and on levels of exposed Dectin-1 protein. Table shows mean values with (standard errors).
AF488 Zymosan
Cell Type
(% Parent Population)		Anti-Dectin-1
Antibody		Isotype Control
Antibody		p Value
CD14+ cells85.47 (1.6)86.63 (0.9)0.33
AF488+ CD14+ cells76.17 (0.6)86.43 (0.7)4.3 × 10−5
AF488− CD14+ cells23.03 (0.8)12.63 (0.6)5.9 × 10−5
Geometric Mean Fluorescence Dectin-1
AF488+ CD14+ cells203.33 (13.1)244.67 (11.2)0.014
AF488− CD14+ cells319.33 (20.7)444.67 (4.5)0.00051
GFP− M. abscessus
Cell Type
(% parent population)		Anti-Dectin-1
Antibody		Isotype Control
Antibody		p value
CD14+ cells88.00 (0.4)87.87 (1.3)0.87
AF488+ CD14+ cells36.27 (2.8)37.90 (2.6)0.49
AF488− CD14+ cells63.03 (2.9)61.30 (2.2)0.46
Geometric Mean Fluorescence Dectin-1
AF488+ CD14+ cells352.87 (16.0)523.33 (8.0)7.9 × 10−5
AF488− CD14+ cells359.67 (9.3)513.33 (16.8)0.00016
Table
Table 2. Effects of laminarin on MDM ingestion of zymosan and M. abscessus and on levels of exposed Dectin-1 protein. Table shows mean values with (standard errors).
Table 2. Effects of laminarin on MDM ingestion of zymosan and M. abscessus and on levels of exposed Dectin-1 protein. Table shows mean values with (standard errors).
AF488 Zymosan
Cell Type
(% Parent Population)		Anti-Dectin-1
Antibody		Isotype Control
Antibody		p Value
CD14+ cells84.50 (2.2)87.87 (2.6)0.15
AF488+ CD14+ cells78.30 (1.9)90.33 (0.3)0.00040
AF488− CD14+ cells21.13 (1.9)9.12 (0.3)0.00044
Geometric Mean Fluorescence Dectin-1
AF488+ CD14+ cells 133.67 (12.0)248.67 (4.9)0.00011
AF488− CD14+ cells231.67 (4.5)443.33 (21.2)7.1 × 10−5
GFP− M. abscessus
Cell Type
(% parent population)		Anti-Dectin-1
Antibody		Isotype Control
Antibody		p value
CD14+ cells85.67 (1.0)88.80 (0.90)0.015
AF488+ CD14+ cells34.30 (1.8)42.70 (2.7)0.011
AF488− CD14+ cells64.57 (1.6)56.37 (2.6)0.0099
Geometric Mean Fluorescence Dectin-1
AF488+ CD14+ cells255.00 (3.0)528.00 (19.5)1.8 × 10−5
AF488− CD14+ cells259.00 (2.7)521.00 (9.6)1.4 × 10−6
Table
Table 3. Relative abundance of immune cell populations in the BAL and lungs of wild-type and Dectin-1−/− mice during acute pulmonary infection with M. abscessus. Table shows mean values with (standard errors).
Table 3. Relative abundance of immune cell populations in the BAL and lungs of wild-type and Dectin-1−/− mice during acute pulmonary infection with M. abscessus. Table shows mean values with (standard errors).
Cell Type
(% Parent Population)		Wild-Type
Mice		Dectin-1−/−
Mice		p Value
BAL
CD45+48.5 (1.0)58.2 (7.1)0.11
PMN19.0 (9.3)17.2 (4.3)0.71
Total macrophages40.9 (20.9)51.5 (5.97)0.29
% Dectin-1+ total macrophages90.0 (3.5)7.1 (1.4)3 × 10−11
Resident macrophages88.2 (3.8)91.9 (2.5)0.11
Recruited macrophages10.5 (4.0)7.0 (2.4)0.12
Lung Homogenate
CD45+73.74 (6.8)72.46 (4.8)0.79
PMN5.61 (1.9)6.18 (0.8)0.50
Total macrophages10.53 (5.2)11.64 (4.7)0.75
Airspace macrophages62.80 (12.4)63.44 (9.8)0.82
Non-airspace macrophages
(recruited and interstitial)		35.30 (12.7)34.72 (9.7)0.84
Table
Table 4. Flow cytometry antibodies used to stain human cells.
Table 4. Flow cytometry antibodies used to stain human cells.
FluorophoreAntibody TargetCloneCompanyCatalog Number
APCCD1461D3Invitrogen
(Waltham, MA, USA)		17-0149-42
PerCP Cy5.5CD369 (Dectin-1)15e2Biolegend
(San Diego, CA, USA)		355408
FITCTLR2TL2.2Invitrogen11.99.22.42
Calcein Blue-AMIntact, viable cellsn/aInvitrogen65-0855 39
Table
Table 5. Flow cytometry antibodies used to stain murine cells.
Table 5. Flow cytometry antibodies used to stain murine cells.
FluorophoreAntibody TargetCloneCompanyCatalog Number
AlexaFluor 405CD4530.F11Invitrogen48045182
FITCLy-6G1A8BD Biosciences
(Franklin Lakes, NJ, USA)		551460
PESiglec-FE50-2440BD Biosciences552126
PECD8820/70Biolegend135806
APCCD8820/70Biolegend135808
PerCP-eFluor 710Clec7a/Dectin-1bg1fpJInvitrogen46-5859-82
PE-Cy7CD64X54-5/7.1Biolegend139314
APC-Cy7CD11cN418Biolegend117324
BUV395Ly-6G1A8BD Biosciences563978
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MDPI and ACS Style

Ochoa, A.E.; Congel, J.H.; Corley, J.M.; Janssen, W.J.; Nick, J.A.; Malcolm, K.C.; Hisert, K.B. Dectin-1-Independent Macrophage Phagocytosis of Mycobacterium abscessus. Int. J. Mol. Sci. 2023, 24, 11062. https://doi.org/10.3390/ijms241311062

AMA Style

Ochoa AE, Congel JH, Corley JM, Janssen WJ, Nick JA, Malcolm KC, Hisert KB. Dectin-1-Independent Macrophage Phagocytosis of Mycobacterium abscessus. International Journal of Molecular Sciences. 2023; 24(13):11062. https://doi.org/10.3390/ijms241311062

Chicago/Turabian Style

Ochoa, Alma E., Jack H. Congel, Jodi M. Corley, William J. Janssen, Jerry A. Nick, Kenneth C. Malcolm, and Katherine B. Hisert. 2023. "Dectin-1-Independent Macrophage Phagocytosis of Mycobacterium abscessus" International Journal of Molecular Sciences 24, no. 13: 11062. https://doi.org/10.3390/ijms241311062

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