Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2012 Jun 15.
Published in final edited form as: J Immunol. 2011 May 9;186(12):7110–7119. doi: 10.4049/jimmunol.1100001

Lung Neutrophils Facilitate Activation of Naïve Antigen Specific CD4+ T Cells During Mycobacterium tuberculosis Infection1

Robert Blomgran *, Joel D Ernst *,†,‡
PMCID: PMC3376160  NIHMSID: NIHMS383759  PMID: 21555529

Abstract

Initiation of the adaptive immune response to Mycobacterium tuberculosis occurs in the lung-draining mediastinal lymph node, and requires transport of M. tuberculosis by migratory dendritic cells (DCs) to the local lymph node. The previously-published observations that: 1) neutrophils are a transiently prominent population of M. tuberculosis-infected cells in the lungs early in infection; and 2) that the peak of infected neutrophils immediately precedes the peak of infected DCs in the lungs, prompted us to characterize the role of neutrophils in the initiation of adaptive immune responses to M. tuberculosis.

We found that, although depletion of neutrophils in vivo increased the frequency of M. tuberculosis infected DCs in the lungs, it decreased trafficking of DCs to the mediastinal lymph node. This resulted in delayed activation (CD69 expression) and proliferation of naïve M. tuberculosis Ag85B-specific CD4 T cells in the mediastinal lymph node. To further characterize the role for neutrophils in DC-migration we used a Transwell chemotaxis system and found that DCs that were directly infected by M. tuberculosis migrated poorly in response to CCL19, an agonist for the chemokine receptor CCR7. In contrast, DCs that had acquired M. tuberculosis through uptake of infected neutrophils exhibited unimpaired migration.

These results reveal a mechanism wherein neutrophils promote adaptive immune responses to M. tuberculosis by delivering M. tuberculosis to DCs in a form that make DCs more effective initiators of naïve CD4 T cell activation. These observations provide insight into a mechanism for neutrophils to facilitate initiation of adaptive immune responses in tuberculosis.

Introduction

Despite the availability of drugs to treat it, tuberculosis (TB) remains a major burden to human health. Mycobacterium tuberculosis infects via inhalation and resides in diverse professional phagocytes in the lungs where it utilizes strategies such as preventing phagosome maturation and subversion of host cell death pathways in order to survive and replicate (1). Effective immunity against M. tuberculosis requires CD4+ Th1 and CD8+ T lymphocyte responses to M. tuberculosis antigens (2–5). Compared to other lower respiratory tract infections such as influenza A (6), where the peak in naïve T cell proliferation occurs 4 days after infection, the onset of the CD4+ response against M. tuberculosis is delayed until 10–12 days after aerosol infection (7–9), giving the bacterium time to expand and establish a niche that allows it to resist eradication.

Polymorphonuclear neutrophils are abundant, motile cells involved in the innate immune response and form an early line of defense against microbial pathogens. These professional phagocytes are crucial in defense against extracellular bacterial and fungal infections. Although parasites such as Leishmania have evolved to exploit neutrophils in order to establish and promote disease (10), neutrophils play a protective role against certain other intracellular pathogens (11–14). In an in vivo intranasal Mycobacterium bovis BCG infection model, neutrophils were suggested to have a dual role in acute infection, a direct antimicrobial activity counterbalanced by anti-inflammatory properties (15). Furthermore, innate immune responses to M. tuberculosis in RAG-deficient mice revealed a compensatory function for neutrophils in keeping the bacterial burden in check in the absence of IFNγ (16). Besides a direct bactericidal or immunomodulatory effect, neutrophils readily undergo apoptosis, and phagocytosed microbe-containing apoptotic neutrophils can have a stimulatory effect on macrophages (17) and on DCs (18). Additionally Davis et al. clearly showed that spread of bacteria through apoptotic cells is a major mechanism by which macrophages obtain virulent mycobacteria in vivo (19). Although neutrophils have been shown to contribute to innate protection against mycobacteria (15, 16, 20–23), data to the contrary are similarly compelling (15, 24–26). Other than the neutrophil’s capacity to produce chemokines/cytokines (27–30), in vivo evidence for a role of neutrophils in modulating adaptive immunity during M. tuberculosis infections has not been reported.

Evidence for one or more roles of neutrophils in human immunity to TB includes the observation that the risk of TB infection among household contacts is inversely associated with peripheral blood neutrophil count, and killing of M. bovis BCG in a whole-blood in vitro assay was significantly impaired by neutrophil depletion (20). Moreover, humans exhibit a transcriptional signature in peripheral blood that indicates a role for neutrophils and/or a related myeloid cell that occurs in response to active pulmonary tuberculosis (31). Consequently, greater understanding of the roles neutrophils play in the innate and adaptive immune responses to M. tuberculosis is needed.

Dendritic cells are potent antigen presenting cells that prime naïve T cells in the lung-draining lymph node (mediastinal lymph node, MDLN) following M. tuberculosis infection (32, 33). Initial activation of naïve M. tuberculosis-specific CD4+ T cells in the MDLN depends on DC transport of bacteria from the lungs to the MDLN (9), in an IL12p40 homodimer- (32) and temporally CCR7-dependent manner (34). Furthermore, when characterizing the cells harboring M. tuberculosis following aerosol infection of mice, we found that neutrophils were a transiently dominant population of lung cells infected early in infection (35). The observation that the peak number of infected neutrophils immediately preceded the peak of infected DCs in the lungs suggests at least two competing hypotheses: 1) acquisition of M. tuberculosis by neutrophils transiently sequesters the bacteria and delays their acquisition by DCs; or 2) infected neutrophils interact with DCs to promote DC acquisition of the bacteria and bacterial antigens. To test these hypotheses and to characterize the role of neutrophils in the initiation of adaptive immune responses to M. tuberculosis, we depleted neutrophils in vivo using a mAb against the neutrophil-specific antigen Ly6G (clone 1A8) (15, 36). We found that neutrophils were necessary for timely initiation of the adaptive immune response by supporting DC migration and trafficking of M. tuberculosis to the local lymph node.

Materials and Methods

Mice

C57BL/6 mice were bred and housed in a specific pathogen-free environment in New York University School of Medicine (New York, NY) animal facilities or purchased from The Jackson Laboratory (Bar Harbor, ME). P25TCR-Tg mice, whose CD4+ T cells express a transgenic T cell Ag receptor that recognizes peptide 25 (aa 240–254) of M. tuberculosis Ag85B bound to I-Ab were on a C57BL/6 background (CD45.2) or on a Rag1−/− background (when specified), as previously described (37), and were bred in the New York University School of Medicine animal facilities. CD45.1 mice were either bred in New York University School of Medicine animal facilities or purchased from Taconic Farms, Inc. Genotypes of mice were confirmed by PCR testing of tail genomic DNA. All procedures conducted on mice were in accordance with the conditions specified by the New York University School of Medicine Institutional Animal Care and Use Committee.

Antibodies, FACS staining and acquisition

All Abs were purchased from BD Pharmingen unless otherwise stated. Anti-CD11c PerCP (H3L) (1:200) was custom conjugated from BD Pharmingen, and other Ab conjugates used were anti-CD45.2 PerCP (1:200), anti-CD4 Alexa Fluor 647 (1:200), anti-CD69 PE (1:200), anti-CD11b PE or PB (1:1500), anti-CD40 Alexa Fluor 647 or PE (1:200), anti-CD86 APC or PE (1:200), anti-CD80 Alexa Fluor 647 or PE (1:200), anti-MHC II Alexa Fluor 647 or PE (1:1500), CCR7 Alexa Fluor 647 or PE (1:200), anti-Ly6C FITC or PE (1:600), anti-Ly6G Alexa Fluor 647 (1:600), Gr-1 APC (1:1500). Staining for surface markers was done by resuspending up to 1×106 cells in 100 μl FACS-buffer (PBS supplemented with 1% heat-inactivated fetal bovine serum, 0.1% NaN3 and 1 mM EDTA) containing Abs and incubated at 4°C for 25 min (or at 37°C for CCR7). Cells were then washed twice and fixed overnight in PBS/1% paraformaldehyde at 4°C. Data were acquired using FACS Calibur or LSR II flow cytometer depending on the experiment.

P25TCR-Tg CD4+ T cell isolation and labeling

P25TCR-Tg mice between 8–16 wk of age were killed according to approved laboratory animal procedures, and naïve P25TCR-Tg CD4+ T cells from lymph nodes and spleen were isolated as previously described (9). For proliferation assays, CD4+ T cells were labeled with CFSE (CFDA-SE; Invitrogen).

Adoptive transfer and aerosol infection

CD45.1 mice routinely received 2–3 × 106 CFSE-labeled P25TCR-Tg CD4+ T cells (CD45.2) by tail vein or retro-orbital injection, in 100 μl of sterile PBS. 3–24 h post cell transfer, mice were infected by the aerosol route using an Inhalation Exposure Unit (Glas-Col), and the infectious dose was confirmed by euthanizing 4–5 mice and plating homogenized lungs within 24 h of infection as previously described (35).

Tissue processing and CFU determination

Mice were euthanized at designated time points, and tissues were used to prepare single-cell suspensions and to determine the bacterial loads by plating, as previously described (9, 35).

Phenotyping and quantitation of lung cells

To avoid epitope masking in mice treated with the neutrophil-depleting antibody to Ly6G, 1A8, neutrophils were defined and quantitated as CD11bhi/Gr-1hi/Ly6Cint/CD11clo/neg (35, 36, 38). For identification of lung macrophage and dendritic cell subsets, neutrophils were first gated out. Based on previous functional and morphological characterization the following lung cell subsets were designated as alveolar macrophages (CD11blow/CD11chigh), myeloid DCs (CD11bhigh/CD11chigh), recruited macrophages (CD11bhigh/CD11cintemediate), and monocytes (CD11bhigh/CD11cnegative)(35, 39).

In vivo neutrophil depletion

The purified Ly6G-specific antibody 1A8 (36) was used to deplete neutrophils in vivo; purified 2A3 (Rat IgG2a) was used as isotype control antibody; both were obtained from BioXcell (West Lebanon, NH). Single dose treatment of 300 μg administered i.p. was used to prevent the confounding effects of an immune response towards the depleting Ab, which can be seen with multiple treatments in vivo (25). Furthermore, 1A8 had no effect on Ly6C+ cells in spleen or lungs two days after administration, at which time neutrophils were fully depleted (our data not shown and (12)).

Bacterial strains, treatment, and in vitro infection

M. tuberculosis (H37Rv) and FACSoptimized GFP-H37Rv (under the control of the Mycobacterium bovis BCG Hsp60 promoter) was prepared for in vivo and in vitro use as previously described (35). For in vitro use log-phase bacteria (OD580 = 0.5–0.9) were washed, resuspended in 15 ng/ml mouse granulocyte macrophage-colony stimulating factor supplemented RPMI-10 complete medium (referred to as GMCSF-medium) and gravity filtered through a 5 μm filter to obtain single cell bacteria. Multiplicity of infection (MOI) was calculated depending on the optical density at 580 nm and validated through serial dilutions and plating. DCs were infected with an MOI=5 for 19 hours yielding 60–70% of the bone marrow DCs (BMDCs) infected. To optimize uptake of M. tuberculosis by neutrophils and synchronize the assay, log-phase GFP-H37Rv bacteria were opsonized using 50% pooled AB human serum in RPMI without additives for 30 min at 37°C, before addition to neutrophils (MOI=5), allowing for 40 min phagocytosis at 37°C (routinely yielding 70–80% GFP+ neutrophils according to flow cytometry). Infected cells were washed thrice, treated for 40 min with 200 μg/ml amikacin and further washed twice before used. Neutrophils were additionally labeled with CMTMR (CellTracker™ Orange; Invitrogen) for selected experiments.

BMDC and neutrophil isolation

Bone marrow from C57BL/6 mice was cultured in GMCSF-medium at 37°C, 5% CO2. Fresh GMCSF-medium was added day 3 and day 6. The floating cell fraction was collected at day 7 and used as source of BMDCs and neutrophils by positive selection using magnetic beads coupled to Anti-CD11c mAb (N418) or Anti-Ly-6G, respectively, and AutoMACS sorted according to the manufacturer’s instructions (Miltenyi Biotec, Auburn, CA, USA). Cells were maintained in GMCSF-medium throughout the experiment to prevent cytokine withdrawal-induced cell death. GMCSF-medium; RPMI-1640 with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 1 mM sodium pyruvate, 1x β-Mercaptoethanol, 10 mM HEPES, and 15 ng/ml mouse GM-CSF; culture supernatants from GM-CSF-producing melanoma cells quantified using mouse GM-CSF ELISA kit from BioSource International, and stored as aliquots in −80°C until use.

Flt3L expanded in vivo DCs

To increase the number of in vivo DCs C57BL/6 mice were subcutaneously injected with 3–6 × 106 fms-like tyrosine kinase 3 ligand (Flt3L)-producing melanoma cells of 80% confluence, and splenocytes were isolated 7–9 days later. Spleens were excised, forced through a 70 μm nylon cell strainer (BD Falcon), and red blood cells were removed using ACK lysis buffer. These in vivo-derived DCs were purified by CD11c positive selection using magnetic beads coupled to anti-CD11c mAb. This resulted in ≥ 96% DC purity. To generate mature DCs, cells were incubated overnight in GMCSF-medium at 37°C, 5% CO2. Mature DCs were CD40hi, CD80hi, CD86hi and major histocompatibility complex (MHC) IIhi. By contrast immature DCs were CD40neg/lo, CD80neg/lo, CD86neg/lo and MHC IIlo. Maturation of DCs was further validated using fluid phase endocytosis of FITC-dextran, where mature DCs routinely showed a 50–75% decreased endocytosis compared to immature DCs. Cellular uptake of FITC-dextran (m.w. 40,000; Sigma St. Louis, MO, USA) was quantified by flow cytometry expressed as the mean fluorescent intensity, where the background fluorescence of cells incubated with FITC-dextran at 4°C was subtracted.

Generation of P25 Rag1−/− effector T cells

P25TCR-Tg CD4+ T cells on a Rag1−/− background were cultured with irradiated splenic APCs in the presence of 0.5 μM peptide 25, 10 ng/ml IL-12p70, 5 ng/ml IL-2, and 50 ng/ml anti-IL-4 for three days. T cells were further cultured in fresh media containing only IL-2 without peptide 25 for the next 4 days. At the time of assay cells were washed and added to DCs in GMCSF-medium without IL-2.

In vitro T cell stimulation assay

BMDCs were plated at 105 cells/well in flat bottom 48-well tissue culture plates and allowed to adhere for 4 hours. Thereafter DCs were infected with M. tuberculosis or stimulated with M. tuberculosis-containing/amikacin-treated neutrophils (1:1; DC:neutrophil). After 19 hours incubation, potential extracellular bacteria were removed by washing with PBS. P25TCR-Tg Rag1−/− effector T cells or CFSE-labeled naïve P25TCR-Tg CD4+ T cells were added at 4 × 105 cells/well in GMCSF-medium (1 ml total volume). For analyzing activation of effector T cells supernatants were collected and sterile filtered (0.22 μm) before assay for IFNγ by ELISA (BD Bioscience). Analysis for proliferation of naïve P25TCR-Tg CD4+ T cells was done by evaluating CFSE dilution by flow cytometry (9, 34).

In vitro migration assay

DCs (1.5×105) were added to upper wells of 5 μm pore size polycarbonate filters in 6.5 mm diameter 24-well Transwell chambers (Corning via Fisher) in 100 μl, with 600 μl chemokine or medium alone (to determine spontaneous migration) in the lower wells. Recombinant mouse MIP-3β/CCL19 was added to a final concentration of 2 nM (R&D System). Migration assays were conducted in GMCSF-medium, for 1 h (Flt3L-derived in vivo DCs) or 2–3 hours (BMDCs); longer times increased the spontaneous migration without affecting directed migration. Thereafter, 60 μl 30 mM EDTA was added to lower wells and further incubated for 5 min at 37°C. The migrated cells recovered from the lower wells were stained with Abs and fixed overnight in PBS/1% paraformaldehyde. Migrated cells and the number of starting cells were counted for 1 min at a constant flow rate with continuous mixing in a FACS Calibur using CellQuest software (BD Biosciences). Each assay was performed in triplicate. The percentage of migrating cells was calculated from the ratio of migrating cells to starting cells.

Statistical analysis

Unless otherwise indicated, statistical comparison was performed by the unpaired, two-tailed Student t test, using Prism 4 for Macintosh (version 4.0a) from GraphPad Software (GraphPad, SanDiego, CA). p Values: *,p <0.05 were considered significant, but **, p<0.01; and ***, p<0.001, are also shown when appropriate.

Results

1A8 efficiently depletes neutrophils in M. tuberculosis-infected mice

We initiated our studies of the role of neutrophils in immune responses to M. tuberculosis by characterizing the kinetics of neutrophil accumulation in the bronchoalveolar lavage fluid (BAL) and lung parenchyma after low dose aerosol infection. We found that there is a small population of neutrophils (1.5–1.7×105 neutrophils/mouse) in the parenchyma on days 1 and 4 post infection. Beginning by day 7, there was a progressive increase in the number of neutrophils in the lung for the duration of the experiments (Fig. 1A). Since the number of neutrophils in the BAL was less than 1% of the total lung PMN at any time point studied, we did not separate BAL and lung parenchymal neutrophils in subsequent experiments. To determine whether neutrophils promote or deter T cell activation during initiation of the adaptive immune response to M. tuberculosis, we depleted neutrophils in C57BL/6 mice 9 days after infection, before the earliest activation of CD4+ T cells is observed (3, 8, 9, 34). First, using the conventional Gr-1high/CD11bhigh gating strategy for neutrophils, which also was confirmed by staining with Gr-1/Ly6C staining (38) (Fig. 1B), we established that a single dose of 1A8 depleted neutrophils (Gr-1high, CD11bhigh) from blood (by 94.3 % ± 3.12%) and lungs (by 89.6% ± 2.76%) in infected mice (mean depletion ± SD; n=3; flow plots for lungs shown in Fig. 1B). Furthermore, we verified that 1A8 showed no epitope masking that would prevent subsequent detection of residual neutrophils by the widely-used antibody to Gr-1 (clone RB6-8C5, which recognizes both Ly6G and Ly6C) (data not shown). Next, we determined that administration of 1A8 on day 9 post-infection caused profound depletion of neutrophils in the lungs until at least day 14, and that neutrophils remained significantly depleted through day 21 (Fig. 1E). No other cell subsets examined were depleted. However, depletion of neutrophils caused a small but significant increase in the number of myeloid DCs, recruited macrophages, and monocytes in the lungs as compared to isotype treated control mice (Fig. 1E), confirming that neutrophils may have a regulatory effect on recruitment of inflammatory cells to the lungs (15). Furthermore, there was no difference in the bacterial burden in the lungs between neutrophil-depleted and neutrophil-replete mice (Fig. 1D), indicating that neutrophils do not contribute to killing of M. tuberculosis during this phase of the infection. This is in accord with Pedrosa et al. who showed that administration of a Ly6G/Ly6C-specific mAb (RB6-8C5) at day 16 post infection with M. tuberculosis, via i.v infection, did not affect the total bacterial burden in liver, spleen, or lungs of BALB/c mice (21).

Figure 1.

Figure 1

Open in a new tab

Myeloid cell and bacterial populations in the lungs of neutrophil depleted and control mice. (A) Neutrophil accumulation in the BAL and lung parenchyma during the early stages of M. tuberculosis infection. (B) Representative flow plots of lung neutrophils at day 17 post infection (when lung neutrophils are abundant), in mice treated with isotype or 1A8 three days earlier. Shown is the gating strategy routinely used to identify neutrophils (PMN) (Gr-1hi/CD11bhi); Gr-1/Ly6C staining was performed to evaluate and confirm the validity of the Gr-1/CD11b gating. Neutrophils were additionally CD11clo/neg (not shown). (C) Experimental scheme for analysis of effects of neutrophil depletion in M. tuberculosis-infected mice. Mice were infected with ~100 CFU/mouse. (D) Lung bacterial burdens in neutrophil-depleted and control mice. No statistically significant differences were observed. (E) Effects of neutrophil depletion on myeloid cell populations in the lungs on days 14, 17, and 21 postinfection. Data are mean ± SD of five mice per group and time point. The results shown in panels D and E are representative data of three separate experiments. PMN, neutrophils; AM, alveolar macrophages; mDC, myeloid dendritic cells; RM, recruited macrophages; Mo, monocytes. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Neutrophil deletion results in cellular redistribution of M. tuberculosis in the lungs

To determine whether neutrophils either delay or accelerate the acquisition of M. tuberculosis by myeloid DCs in the lungs, mice were treated with 1A8 or isotype control at day 16 post-infection and analyzed at day 19. This time point was chosen since it overlaps with the peak of infected neutrophils in the lungs of untreated mice (35), and because at earlier time points few myeloid DCs are present in the lungs. We used M. tuberculosis expressing FACS-optimized GFP to identify the infected cells by flow cytometry. With depletion of neutrophils there was a 2-fold increase in the total number of infected DCs (Fig. 2A). This redistribution of M. tuberculosis was not specific to DCs, since recruited macrophages and monocytes showed similar increases, while alveolar macrophages did not. Furthermore, when considering all of the infected lung cell subsets, ≥ 98% of the GFP+ events in the control group were accounted for in the 1A8-treated group. Quantitation of M. tuberculosis CFU in tissue homogenates revealed no difference in bacterial loads in lungs of neutrophil depleted vs. isotype treated control mice when treated at day 16 and analyzed at day 21 post infection (Fig. 2B).

Figure 2.

Figure 2

Open in a new tab

Neutrophil depletion redistributes M. tuberculosis to other myeloid cells in the lungs. C57BL/6 mice aerosol infected with a low dose of GFP-expressing H37Rv were treated with 1A8 or isotype control antibody at day 16 post infection and analyzed at day 19 (A) or day 21 (B). No difference in the bacterial burden in lung or MDLN between groups was detected. Data are mean ± SD of five mice per group and time point. PMN, neutrophils; AM, alveolar macrophages; mDC, myeloid dendritic cells; RM, recruited macrophages; Mo, monocytes. **, p < 0.01.

Neutrophils promote timely proliferation of M. tuberculosis Ag85B-specific CD4+ T cells

We previously reported that proliferation of naïve P25TCR-Tg CD4+ T cells occurs in the MDLN (and not the lungs), and that this initiation of the adaptive immune response to M. tuberculosis Ag85B requires transport of live bacteria from the lungs to the local draining lymph node (9). To characterize the timing and dependence on neutrophils in activation of naïve M. tuberculosis specific T cells, we adoptively transferred CFSE labeled P25TCR-Tg CD4+ T (CD45.2) cells into C57BL/6 mice (CD45.1) prior to infection with M. tuberculosis. Mice were treated with 1A8 or isotype control at day 9, and P25TCR-Tg T cells in the lung draining lymph node were analyzed for CD69 expression as well as proliferation at day 14, 15, and 17 post-infection. No evidence of T cell proliferation was detected in either group at day 14, however there was increased expression of the activation marker CD69 in the isotype versus the 1A8 treated group (40.2% ± 5.2% and 20.7 ± 3.5% of adoptively-transferred cells, respectively; p=0.0223) (top left panel of Fig. 3A, and Fig. 3B), indicating earlier activation of lymph node CD4+ T cells in the presence of lung neutrophils. At day 15, isotype treated mice showed a significant increase in P25TCR-Tg CD4+ T cells that had undergone at least one cycle of proliferation compared to that in neutrophil-depleted mice (top right panel of Fig. 3A, and Fig. 3C). By day 17 post infection, active proliferation of CD4+ T cells was observed in neutrophil-depleted mice, and there was no difference in CD4+ T cell proliferation between the groups, indicating that neutrophil depletion delays rather than prevents M. tuberculosis specific T cell responses.

Figure 3.

Figure 3

Open in a new tab

Neutrophils accelerate activation of naïve M. tuberculosis Ag85B-specific CD4+ T cells in vivo. Mice were treated as shown in Figure 1C after adoptive transfer of naïve P25TCR-Tg CD4+ T cells on the day preceding infection. (A) Representative CFSE-dilution profiles of P25TCR-Tg CD4+ T cells in MDLN on days 14, 15, and 17 postinfection. (B) P25TCR-Tg cell activation as reflected by CD69 expression; gating used is shown in panel A. (C) Comparison of P25TCR-Tg CD4+ T cell proliferation in neutrophil-depleted and control mice; gating used is shown in panel A. Data in panels B and C are mean ± SD of five mice per group and time point. Data are representative of two separate experiments. **, p < 0.01.

Neutrophils promote trafficking of M. tuberculosis from lungs to the mediastinal lymph node

Although neutrophils were essential for timely activation of naïve T cell in the MDLN, we did not observe neutrophils (Gr-1high/CD11bhigh) in the lymph node at any time point studied, suggesting that the effects of neutrophils that promote naïve T cell activation occur in the lungs. Since we (9) and others (7), have found that activation of M. tuberculosis-specific T cells is preceded by transport of live bacteria to the lymph node, we examined the effect of neutrophil depletion on trafficking of bacteria to the lymph node. This revealed approximately 2 fold more M. tuberculosis CFU in lymph nodes of neutrophil-replete compared with neutrophil-depleted mice at days 14 and 15, respectively (Fig. 4A), in the absence of a difference in lung CFU at any time point examined (Fig. 1D). We previously found that 1,200–1,500 M. tuberculosis CFU appeared to represent a threshold for promoting proliferation of P25TCR-Tg CD4+ T cells in the mediastinal lymph node (9). The present data (Figs 3C and 4A) are consistent with this, and indicate that neutrophil-replete mice reach this threshold sooner then neutrophil-depleted mice (Fig. 4A). Finally, despite the greater number of myeloid DCs in lungs of neutrophil-depleted animals (Fig. 1E), trafficking of DCs to the MDLN was actually delayed by neutrophil depletion (Fig. 4B). In accord with the presence of fewer bacteria and DCs in the lymph node in the absence of neutrophils, it was not surprising to find that the number of M. tuberculosis-infected (GFP+) cells among the myeloid DC (CD11bhigh/CD11chigh) population in the MDLN was also decreased on day 15 in the neutrophil depleted group (941.2 ± 226 vs. 480.1 ± 87.47 in neutrophil-replete and neutrophil-depleted mice, respectively; p = 0.0468; n = 5). In contrast to the reduced number of CD11bhigh/CD11chigh myeloid DCs in the MDLN, the number of CD11blow and CD11bneg DCs in the MDLN were unaffected by neutrophil depletion with 1A8 at any time point studied (data not shown). These data together indicate that neutrophils are important for timely acquisition and trafficking of DCs containing M. tuberculosis from the lungs to the MDLN.

Figure 4.

Figure 4

Open in a new tab

Neutrophils accelerate trafficking of M. tuberculosis and dendritic cells from the lungs to the mediastinal lymph node. Mice were treated as shown in Figure 1C. (A) M. tuberculosis CFU in MDLN homogenates at the indicated time-points. (B) CD11bhigh/CD11chigh myeloid dendritic cells on days 14 and 15 postinfection. Data are mean ± SD of five mice per group and time point, representating data from two (A) or one (B) separate experiments. Data in B were analyzed using unpaired one-tailed Student t test. *, p < 0.05.

M. tuberculosis-infected neutrophils efficiently provide antigens to dendritic cells for activation of CD4+ T cells

Since the peak population of M. tuberculosis-infected neutrophils precedes that of infected DCs in the lungs (35), and since depletion of lung neutrophils delayed activation of CD4+ T cells in the lymph node, we considered the possibility that M. tuberculosis-infected neutrophils interact with DCs in the lungs in a manner that promotes subsequent activation of naïve CD4+ T cells. We first examined the possibility that infected neutrophils can provide M. tuberculosis antigens to DCs for presentation to CD4 T cells in vitro. To compare the ability of DCs to present M. tuberculosis Ag85B after direct infection or after the bacteria had first been ingested by neutrophils, we provided BMDCs with M. tuberculosis directly, or with the same number of bacteria contained in neutrophils, and then assayed the activation of P25TCR-Tg CD4+ effector T cells. In particular, we quantitated effector T-cell activation as IFNγ secretion after 6, 12, 24, and 36 hours of coincubation. This revealed that activation of antigen-specific CD4+ T cells was as efficient when DCs were provided bacteria contained in neutrophils as when the DCs acquired bacteria directly (Fig. 5A). These results indicate that DC uptake of M. tuberculosis-infected neutrophils results in efficient presentation of bacterial antigens. To further characterize the presentation of M. tuberculosis antigens after neutrophil infection, we determined whether the ability of DCs to acquire and present antigens from infected neutrophils also occurred at lower MOI’s. This revealed that DCs that acquired M. tuberculosis antigen from infected neutrophils activated CD4+ T cells as effectively as directly-infected DCs at all MOI examined (Fig. 5B). Next, we examined the ability of DCs to stimulate proliferation of naïve Ag85B-specific CD4+ T cells after incubation with infected neutrophils or with M. tuberculosis alone. This revealed that DCs that contained bacteria acquired from infected neutrophils were as efficient at inducing proliferation of naïve P25TCR-Tg cells as DCs that contained bacteria acquired by direct infection (Fig. 5C). These data indicate that antigen presentation by DCs is as efficacious if M. tuberculosis antigens are acquired from infected neutrophils or obtained by direct infection.

Figure 5.

Figure 5

Open in a new tab

M. tuberculosis -infected neutrophils provide antigen to dendritic cells for presentation to CD4+ T cells. (A) BMDCs were directly infected with M. tuberculosis (Mtb; MOI=5) or incubated with an equal number of neutrophils that were previously infected with M. tuberculosis (MOI=5) for 19 hours. Thereafter, BMDCs were incubated with P25TCR-Tg Th1 effector CD4+ T cells, and release of IFNγ into supernatants was analyzed after 6, 12, 24 and 36 hours. (B) BMDCs were directly infected with M. tuberculosis at the MOI shown or incubated with an equal number of M. tuberculosis-infected neutrophils (previously infected at the MOI shown), then incubated with P25TCR-Tg Th1 effector CD4+ T cells, and release of IFNγ into supernatants was analyzed after 12 hours. (C) CFSE-labeled naïve P25TCR-Tg CD4+ T cells were added to BMDC that were either directly infected or after incubation with infected neutrophils as described for panels 5A and B, and T-cell proliferation was analyzed at 72 hours. Mean values of cells that had undergone at least one cycle of proliferation are depicted in histograms. Data in A–C are representative of two separate experiments performed in triplicate. Un = undetected.

Acquisition of bacteria from infected neutrophils prevents M. tuberculosis inhibition of DC migration

Since we observed that depletion of neutrophils delayed DC migration and trafficking of M. tuberculosis from the lungs to the MDLN, we hypothesized that neutrophils promote migration of DCs in the context of M. tuberculosis infection. Such a hypothetical effect could operate by directly promoting migration or by abrogating a negative effect of M. tuberculosis on migration. Therefore, we determined whether acquiring bacteria through distinct routes (either directly or by ingesting infected neutrophils) affects the migratory properties of DCs in response to CCL19, a chemoattractant for mature DCs. To compare migration of infected and uninfected DCs from a mixed population, we used GFP-expressing M. tuberculosis and flow cytometry to distinguish infected and uninfected cells in the migration assay. We also used CMTMR labeled neutrophils to identify DCs that had ingested neutrophils (Fig. 6A). The upper panel of figure 6A shows the gating strategy excluding extracellular neutrophils (Ly6G+), and R1–R4 in figure 6B indicates the quadrant of the lower panel in figure 6A that is analyzed for migration. Approximately 20% of the uninfected control cells as well as the DCs containing infected neutrophils (R2; Ly6G−/CD11c+/GFP+/CMTMR+) migrated in response to CCL19, whereas approximately 7% of the DCs directly infected with M. tuberculosis (R4; CD11c+/GFP+) migrated to the lower chamber during the three-hour assay. The decreased migration of directly-infected DCs was associated with decreased expression of the chemokine receptor, CCR7, compared with that expressed by DCs that contained M. tuberculosis-infected neutrophils (Fig. 6C). This indicates that direct M. tuberculosis infection of DCs causes a 2.4-fold decrease in migration compared to that of uninfected cells, and that this inhibitory effect of M. tuberculosis is absent when DCs acquire M. tuberculosis by ingesting infected neutrophils. These data indicate that although antigen presentation and activation is equivalent between the groups, DCs that have acquired M. tuberculosis through neutrophils are superior at migrating towards lymph node chemokines, when compared to directly infected DCs.

Figure 6.

Figure 6

Open in a new tab

M. tuberculosis inhibits DC migration when DCs acquire bacteria directly, but not when they acquire bacteria by ingesting infected neutrophils. Before migration assays, DCs were directly infected with GFP-M. tuberculosis (MOI=5), or incubated with an equal number of either uninfected or infected (MOI=5) CMTMR-labeled neutrophils for 19 hours. To distinguish migration of dendritic cells that contained bacteria, neutrophils, both, or neither, input cells and cells in the lower well of the Transwell chamber after migration were stained with antibodies to CD11c and Ly6G, counted, and analyzed by flow cytometry. (A) Shows the gating strategy used to exclude extracellular neutrophils (Ly6G+; top panel), and R1–R4 (bottom panel) distinguish DCs that contain M. tuberculosis (GFP+), infected neutrophils (CMTMR+/GFP+), or uninfected neutrophils (CMTMR+/GFP−) (BMDCs used as input cells are displayed). (B) BMDCs were allowed to migrate 3 hours in the presence of CCL19. (C) CCR7 expression on uninfected DCs, directly-infected DCs, and DCs that acquired M. tuberculosis by ingesting infected neutrophils. (D) In vivo expanded spleen DCs (Flt3L-DC) did not ingest neutrophils, but could be infected by M. tuberculosis, restricting the evaluation of Flt3L-DC migration to cells incubated with M. tuberculosis (containing bacteria or not) and untreated DCs. In addition to GFP+ DCs, GFP− DCs from the M. tuberculosis stimulated group were also analyzed. Unstimulated CCR7−/− Flt3L-DCs were used to determine baseline random migration. Data are expressed as mean ± SD percent of the input cells that migrated to the lower chamber after 1 hour. Each condition was assayed in triplicate; data are representative of two (B and C) and three (D) independent experiments. Mtb, M. tuberculosis; infPMN, M. tuberculosis-infected neutrophils; uninfPMN, uninfected neutrophils. *, p < 0.05; **, p < 0.01.

We also examined the effect of M. tuberculosis on migration of in vivo-derived CD11b+/CD11c+ spleen dendritic cells. Flt3L-expanded in vivo DCs were more mature than BMDCs, making them ideal for migration studies. However, as one manifestation of their mature phenotype, these cells did not ingest neutrophils, thereby restricting our study to examining the effects of direct infection with M. tuberculosis on migration. As we observed with BMDCs, a lower fraction of in vivo-generated DCs migrated in response to CCL19 if they contained M. tuberculosis (GFP+ DCs) than if they did not contain bacteria (Fig. 6D). As we found with bone marrow-derived DCs, direct infection of Flt3L-expanded DCs by M. tuberculosis decreased expression of CCR7 (data not shown). Of note, the GFP− (non-infected) DCs from the M. tuberculosis treated sample exhibited a 2.4-fold increase in migration compared to that of GFP+ DCs (p=0.0037), as well as a 1.4-fold increase in migration compared to DCs that were not exposed to M. tuberculosis (p=0.0425).

M. tuberculosis infected neutrophils release a chemoattractant for dendritic cells

To further investigate the interaction of neutrophils and DCs, we tested the hypothesis that M. tuberculosis-infected neutrophils release factors that attract DCs in order to facilitate the interactions of these cells in the lungs. We examined culture supernatants from uninfected and M. tuberculosis-infected neutrophils for their capacity to attract previously untreated BMDCs. After infection, neutrophils were extensively washed, amikacin-treated, and incubated for 19 h in fresh media. Cleared supernatants were filtered through a 0.22 μm filter before use in the lower wells of the Transwell® migration system. Medium from uninfected neutrophils did not enhance DC migration above the level of spontaneous migration (with medium alone), whereas supernatants from infected neutrophils increased DC migration to levels comparable to 2 nM CCL19 (Fig. 7). When we used CCR7−/− BMDCs in the assay, there was no detectable difference in migration toward supernatant from uninfected neutrophils versus supernatant from infected neutrophils (not shown), indicating that the chemotactic activity produced by M. tuberculosis-infected neutrophils is attributable to one or more CCR7 agonists, either CCL19 or one of the isoforms of CCL21. This is consistent with previous evidence showing that human neutrophils release MIP-3β(CCL19) when stimulated with either TNF-α, LPS, or gram-positive or gram-negative bacteria (40, 41).

Figure 7.

Figure 7

Open in a new tab

M. tuberculosis -infected neutrophils release one or more chemoattractants for DCs. Particle free and sterile filtered medium from uninfected neutrophils (o/n PMN-sup) or M. tuberculosis infected neutrophils (o/n infPMN-sup) were used as chemotactic stimuli in the lower chamber of Transwell chambers. Data are expressed as mean ± SD percent of the input cells that migrated to the lower chamber after 3 hours. Each condition was performed in triplicate; data are representative of three independent experiments. **, p < 0.01.

Discussion

The findings reported here indicate that neutrophils contribute to activation of antigen-specific CD4+ T cells in response to M. tuberculosis in vivo. Using the Ly6G-specific antibody 1A8 to selectively deplete neutrophils, we found that neutrophils are necessary for optimal trafficking of M. tuberculosis from the lungs to the MDLN, promoting activation and proliferation of M. tuberculosis-specific CD4+ T cells. We further found that DCs that have acquired M. tuberculosis through ingestion of neutrophils that contain bacteria are superior in migrating towards lymph node chemokines when compared to directly-infected DCs. Since we could not detect neutrophils (Gr-1high/CD11bhigh) in the MDLN at any time point studied, our data are most consistent with a model in which infected lung neutrophils convey M. tuberculosis to migratory DCs in a manner that bypasses inhibitory mechanisms of M. tuberculosis, ultimately supporting trafficking of M. tuberculosis-containing DCs to the MDLN and subsequent T cell activation. Our in vivo results confirm and extend prior in vitro observations that M. bovis BCG-infected neutrophils cooperate with DCs to result in DC maturation and effective antigen presentation to CD4 and CD8 T cells (42).

Several recent studies have provided considerable insight into the process and mechanisms of activation of naïve antigen-specific T lymphocytes after infection with M. tuberculosis. These studies have revealed that T cell activation occurs earliest in the lymph node downstream of the lungs, that T cell activation follows the appearance of live M. tuberculosis in the lymph node, and that live bacteria do not reach the lymph node until 8–10 days following aerosol infection (7, 9, 35). Moreover, recent studies have established that myeloid DCs are responsible for transporting M. tuberculosis from the lungs to the lymph node (32, 35), indicating that these cells play a key role in initiating adaptive immune responses to M. tuberculosis after aerosol infection. The data reported here that neutrophils promote activation of antigen-specific CD4 T cells in tuberculosis by facilitating DC migration and antigen presentation provides additional insight into the cellular events that contribute to development of protective immunity in tuberculosis.

Several recent studies have implicated neutrophils in contributing to immune responses in human tuberculosis. In particular, a study of contacts of active TB cases revealed that the risk of TB infection in contacts was inversely proportional to peripheral blood neutrophil counts, and neutrophils contributed to the killing of M. bovis BCG in a whole blood assay (20). Our results in mice suggest that, in addition to a potential direct role in limiting infection, neutrophils contribute to development of an efficacious adaptive immune response. More recently, studies of gene expression in peripheral blood cells of subjects with active tuberculosis and a subset of those with latent tuberculosis infection revealed prominent expression of myeloid genes, especially neutrophils, further indicating a role for these cells in response to M. tuberculosis infection (31). Finally, ex vivo examination of samples of human bronchial lavage and tuberculous cavity contents has revealed the association of a high proportion of acid fast-stained M. tuberculosis with neutrophils in advanced stages of infection (43).

Historically, neutrophil depletion studies in M. tuberculosis infected animals have focused on whether neutrophils have a bactericidal effect or not. For example, early (i.e. during the first week of infection) administration of the neutrophil-depleting antibody RB6-8C5 (which recognizes both Ly6G on neutrophils and Ly6C on certain mononuclear cells) to mice infected intravenously with a high dose of M. tuberculosis resulted in higher bacterial burdens in liver, spleen, and lungs, whereas late administration of RB6 had no effect on bacterial burden in any organ (21). Neutrophils may also contribute to local cytokine production (27–30), to cellular recruitment and granuloma formation (44, 45), and optimal control of virulent M. marinum infection in zebrafish embryos (46).

Although the role of neutrophils in early activation of M. tuberculosis-specific T cells in vivo has not been studied previously, involvement of neutrophils in pathogen-specific adaptive immune responses has been found for other intracellular pathogens. In the case of Leishmania, neutrophil depletion attenuates infection after transmission by sandflies (10), and promotes the development of parasite-specific immune responses in mice vaccinated with heat-killed L. major antigen (47). Therefore, Leishmania exploits neutrophils to promote infection and to limit the efficacy of adaptive immune responses. In contrast, Listeria monocytogenes-infected neutrophils have been found to serve as substrates for dendritic cell cross-presentation of both secreted and nonsecreted antigens in vitro as well as for in vivo cross-priming of CD8+ T cells. Neutrophil depletion also decreased CD8+ T cell responses against nonsecreted Listeria monocytogenes antigens in vivo (48). In Salmonella-infected CCR6-deficient mice, adoptive transfer of wild type blood neutrophils (Gr1+/CD11b+) restored T cell expansion and cell division of CFSE-labeled CD4+ T cells in the draining lymph node (49). Therefore, as in our studies, neutrophils may also contribute to the timely and optimal development of adaptive immune responses and thereby contribute to host protection against other intracellular pathogens.

In summary, we have found that in mice infected with virulent M. tuberculosis by the aerosol route, neutrophils contribute to initial activation of antigen-specific CD4 T cells by cooperating with DCs in the lungs. Our results suggest that DCs acquire M. tuberculosis antigens by ingesting apoptotic neutrophils, indicating that inhibition of neutrophil apoptosis may contribute to the virulence of M. tuberculosis (50–54). Future studies of the roles of neutrophils in defense against M. tuberculosis are likely to reveal additional functions of these cells, which have been traditionally considered to function in innate immunity, but whose roles in modulating adaptive immune responses to intracellular pathogens are becoming more evident.

Acknowledgments

We thank Dr. Adrian Erlebacher and Mary Collins for CCR7−/− mice. We also thank Tawania Fergus for excellent technical assistance, and to Sofia Olmos and Smita Srivastava for advice and assistance at early stages of the project.

Abbreviations

BMDC

bone marrow dendritic cell

DC

dendritic cell

MDLN

mediastinal lymph node

MOI

multiplicity of infection

TB

tuberculosis

Footnotes

1

This work was supported by a grant from the NIH (R01 AI51242) and by the Fulbright Commission in Sweden visiting scholarship, the Swedish Heart Lung Foundation, and the Swedish Research Council.

References

  • 1.Meena LS, Rajni Survival mechanisms of pathogenic Mycobacterium tuberculosis H37Rv. FEBS J. 2010;277:2416–2427. doi: 10.1111/j.1742-4658.2010.07666.x. [DOI] [PubMed] [Google Scholar]
  • 2.Mogues T, Goodrich ME, Ryan L, LaCourse R, North RJ. The relative importance of T cell subsets in immunity and immunopathology of airborne Mycobacterium tuberculosis infection in mice. J Exp Med. 2001;193:271–280. doi: 10.1084/jem.193.3.271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Gallegos AM, Pamer EG, Glickman MS. Delayed protection by ESAT-6-specific effector CD4+ T cells after airborne M. tuberculosis infection. J Exp Med. 2008;205:2359–2368. doi: 10.1084/jem.20080353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Hoang TT, Nansen A, Roy S, Billeskov R, Aagaard C, Elvang T, Dietrich J, Andersen P. Distinct differences in the expansion and phenotype of TB10.4 specific CD8 and CD4 T cells after infection with Mycobacterium tuberculosis. PLoS One. 2009;4:e5928. doi: 10.1371/journal.pone.0005928. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Woodworth JS, Behar SM. Mycobacterium tuberculosis-specific CD8+ T cells and their role in immunity. Crit Rev Immunol. 2006;26:317–352. doi: 10.1615/critrevimmunol.v26.i4.30. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Roman E, Miller E, Harmsen A, Wiley J, Von Andrian UH, Huston G, Swain SL. CD4 effector T cell subsets in the response to influenza: heterogeneity, migration, and function. J Exp Med. 2002;196:957–968. doi: 10.1084/jem.20021052. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Chackerian AA, Alt JM, Perera TV, Dascher CC, Behar SM. Dissemination of Mycobacterium tuberculosis is influenced by host factors and precedes the initiation of T-cell immunity. Infect Immun. 2002;70:4501–4509. doi: 10.1128/IAI.70.8.4501-4509.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Reiley WW, Calayag MD, Wittmer ST, Huntington JL, Pearl JE, Fountain JJ, Martino CA, Roberts AD, Cooper AM, Winslow GM, Woodland DL. ESAT-6-specific CD4 T cell responses to aerosol Mycobacterium tuberculosis infection are initiated in the mediastinal lymph nodes. Proc Natl Acad Sci U S A. 2008;105:10961–10966. doi: 10.1073/pnas.0801496105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Wolf AJ, Desvignes L, Linas B, Banaiee N, Tamura T, Takatsu K, Ernst JD. Initiation of the adaptive immune response to Mycobacterium tuberculosis depends on antigen production in the local lymph node, not the lungs. J Exp Med. 2008;205:105–115. doi: 10.1084/jem.20071367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Peters NC, Egen JG, Secundino N, Debrabant A, Kimblin N, Kamhawi S, Lawyer P, Fay MP, Germain RN, Sacks D. In vivo imaging reveals an essential role for neutrophils in leishmaniasis transmitted by sand flies. Science. 2008;321:970–974. doi: 10.1126/science.1159194. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Sayles PC, Johnson LL. Exacerbation of toxoplasmosis in neutrophil-depleted mice. Nat Immun. 1996;15:249–258. [PubMed] [Google Scholar]
  • 12.Appelberg R, Castro AG, Silva MT. Neutrophils as effector cells of T-cell-mediated, acquired immunity in murine listeriosis. Immunology. 1994;83:302–307. [PMC free article] [PubMed] [Google Scholar]
  • 13.Conlan JW. Critical roles of neutrophils in host defense against experimental systemic infections of mice by Listeria monocytogenes, Salmonella typhimurium, and Yersinia enterocolitica. Infect Immun. 1997;65:630–635. doi: 10.1128/iai.65.2.630-635.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Sjostedt A, Conlan JW, North RJ. Neutrophils are critical for host defense against primary infection with the facultative intracellular bacterium Francisella tularensis in mice and participate in defense against reinfection. Infect Immun. 1994;62:2779–2783. doi: 10.1128/iai.62.7.2779-2783.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Zhang X, Majlessi L, Deriaud E, Leclerc C, Lo-Man R. Coactivation of Syk kinase and MyD88 adaptor protein pathways by bacteria promotes regulatory properties of neutrophils. Immunity. 2009;31:761–771. doi: 10.1016/j.immuni.2009.09.016. [DOI] [PubMed] [Google Scholar]
  • 16.Feng CG, Kaviratne M, Rothfuchs AG, Cheever A, Hieny S, Young HA, Wynn TA, Sher A. NK cell-derived IFN-gamma differentially regulates innate resistance and neutrophil response in T cell-deficient hosts infected with Mycobacterium tuberculosis. J Immunol. 2006;177:7086–7093. doi: 10.4049/jimmunol.177.10.7086. [DOI] [PubMed] [Google Scholar]
  • 17.Zheng L, He M, Long M, Blomgran R, Stendahl O. Pathogen-induced apoptotic neutrophils express heat shock proteins and elicit activation of human macrophages. J Immunol. 2004;173:6319–6326. doi: 10.4049/jimmunol.173.10.6319. [DOI] [PubMed] [Google Scholar]
  • 18.Aleman M, de la Barrera S, Schierloh P, Yokobori N, Baldini M, Musella R, Abbate E, Sasiain M. Spontaneous or Mycobacterium tuberculosis-induced apoptotic neutrophils exert opposite effects on the dendritic cell-mediated immune response. Eur J Immunol. 2007;37:1524–1537. doi: 10.1002/eji.200636771. [DOI] [PubMed] [Google Scholar]
  • 19.Davis JM, Ramakrishnan L. The role of the granuloma in expansion and dissemination of early tuberculous infection. Cell. 2009;136:37–49. doi: 10.1016/j.cell.2008.11.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Martineau AR, Newton SM, Wilkinson KA, Kampmann B, Hall BM, Nawroly N, Packe GE, Davidson RN, Griffiths CJ, Wilkinson RJ. Neutrophil-mediated innate immune resistance to mycobacteria. J Clin Invest. 2007;117:1988–1994. doi: 10.1172/JCI31097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Pedrosa J, Saunders BM, Appelberg R, Orme IM, Silva MT, Cooper AM. Neutrophils play a protective nonphagocytic role in systemic Mycobacterium tuberculosis infection of mice. Infect Immun. 2000;68:577–583. doi: 10.1128/iai.68.2.577-583.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Fulton SA, Reba SM, Martin TD, Boom WH. Neutrophil-mediated mycobacteriocidal immunity in the lung during Mycobacterium bovis BCG infection in C57BL/6 mice. Infect Immun. 2002;70:5322–5327. doi: 10.1128/IAI.70.9.5322-5327.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Appelberg R, Castro AG, Gomes S, Pedrosa J, Silva MT. Susceptibility of beige mice to Mycobacterium avium: role of neutrophils. Infect Immun. 1995;63:3381–3387. doi: 10.1128/iai.63.9.3381-3387.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Eruslanov EB, I, Lyadova V, Kondratieva TK, Majorov KB, Scheglov IV, Orlova MO, Apt AS. Neutrophil responses to Mycobacterium tuberculosis infection in genetically susceptible and resistant mice. Infect Immun. 2005;73:1744–1753. doi: 10.1128/IAI.73.3.1744-1753.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Seiler P, Aichele P, Raupach B, Odermatt B, Steinhoff U, Kaufmann SH. Rapid neutrophil response controls fast-replicating intracellular bacteria but not slow-replicating Mycobacterium tuberculosis. J Infect Dis. 2000;181:671–680. doi: 10.1086/315278. [DOI] [PubMed] [Google Scholar]
  • 26.Denis M. Human neutrophils, activated with cytokines or not, do not kill virulent Mycobacterium tuberculosis. J Infect Dis. 1991;163:919–920. doi: 10.1093/infdis/163.4.919. [DOI] [PubMed] [Google Scholar]
  • 27.Cassatella MA. The production of cytokines by polymorphonuclear neutrophils. Immunol Today. 1995;16:21–26. doi: 10.1016/0167-5699(95)80066-2. [DOI] [PubMed] [Google Scholar]
  • 28.Petrofsky M, Bermudez LE. Neutrophils from Mycobacterium avium-infected mice produce TNF-alpha, IL-12, and IL-1 beta and have a putative role in early host response. Clin Immunol. 1999;91:354–358. doi: 10.1006/clim.1999.4709. [DOI] [PubMed] [Google Scholar]
  • 29.Riedel DD, Kaufmann SH. Chemokine secretion by human polymorphonuclear granulocytes after stimulation with Mycobacterium tuberculosis and lipoarabinomannan. Infect Immun. 1997;65:4620–4623. doi: 10.1128/iai.65.11.4620-4623.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Scapini P, Lapinet-Vera JA, Gasperini S, Calzetti F, Bazzoni F, Cassatella MA. The neutrophil as a cellular source of chemokines. Immunol Rev. 2000;177:195–203. doi: 10.1034/j.1600-065x.2000.17706.x. [DOI] [PubMed] [Google Scholar]
  • 31.Berry MP, Graham CM, McNab FW, Xu Z, Bloch SA, Oni T, Wilkinson KA, Banchereau R, Skinner J, Wilkinson RJ, Quinn C, Blankenship D, Dhawan R, Cush JJ, Mejias A, Ramilo O, Kon OM, Pascual V, Banchereau J, Chaussabel D, O’Garra A. An interferon-inducible neutrophil-driven blood transcriptional signature in human tuberculosis. Nature. 2010;466:973–977. doi: 10.1038/nature09247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Khader SA, Partida-Sanchez S, Bell G, Jelley-Gibbs DM, Swain S, Pearl JE, Ghilardi N, Desauvage FJ, Lund FE, Cooper AM. Interleukin 12p40 is required for dendritic cell migration and T cell priming after Mycobacterium tuberculosis infection. J Exp Med. 2006;203:1805–1815. doi: 10.1084/jem.20052545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Tian T, Woodworth J, Skold M, Behar SM. In vivo depletion of CD11c+ cells delays the CD4+ T cell response to Mycobacterium tuberculosis and exacerbates the outcome of infection. J Immunol. 2005;175:3268–3272. doi: 10.4049/jimmunol.175.5.3268. [DOI] [PubMed] [Google Scholar]
  • 34.Olmos S, Stukes S, Ernst JD. Ectopic activation of Mycobacterium tuberculosis-specific CD4+ T cells in lungs of CCR7−/− mice. J Immunol. 2010;184:895–901. doi: 10.4049/jimmunol.0901230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Wolf AJ, Linas B, Trevejo-Nunez GJ, Kincaid E, Tamura T, Takatsu K, Ernst JD. Mycobacterium tuberculosis infects dendritic cells with high frequency and impairs their function in vivo. J Immunol. 2007;179:2509–2519. doi: 10.4049/jimmunol.179.4.2509. [DOI] [PubMed] [Google Scholar]
  • 36.Daley JM, Thomay AA, Connolly MD, Reichner JS, Albina JE. Use of Ly6G-specific monoclonal antibody to deplete neutrophils in mice. J Leukoc Biol. 2008;83:64–70. doi: 10.1189/jlb.0407247. [DOI] [PubMed] [Google Scholar]
  • 37.Tamura T, Ariga H, Kinashi T, Uehara S, Kikuchi T, Nakada M, Tokunaga T, Xu W, Kariyone A, Saito T, Kitamura T, Maxwell G, Takaki S, Takatsu K. The role of antigenic peptide in CD4+ T helper phenotype development in a T cell receptor transgenic model. Int Immunol. 2004;16:1691–1699. doi: 10.1093/intimm/dxh170. [DOI] [PubMed] [Google Scholar]
  • 38.Sawanobori Y, Ueha S, Kurachi M, Shimaoka T, Talmadge JE, Abe J, Shono Y, Kitabatake M, Kakimi K, Mukaida N, Matsushima K. Chemokine-mediated rapid turnover of myeloid-derived suppressor cells in tumor-bearing mice. Blood. 2008;111:5457–5466. doi: 10.1182/blood-2008-01-136895. [DOI] [PubMed] [Google Scholar]
  • 39.Gonzalez-Juarrero M, Shim TS, Kipnis A, Junqueira-Kipnis AP, Orme IM. Dynamics of macrophage cell populations during murine pulmonary tuberculosis. J Immunol. 2003;171:3128–3135. doi: 10.4049/jimmunol.171.6.3128. [DOI] [PubMed] [Google Scholar]
  • 40.Scapini P, Laudanna C, Pinardi C, Allavena P, Mantovani A, Sozzani S, Cassatella MA. Neutrophils produce biologically active macrophage inflammatory protein-3alpha (MIP-3alpha)/CCL20 and MIP-3beta/CCL19. Eur J Immunol. 2001;31:1981–1988. doi: 10.1002/1521-4141(200107)31:7&#60;1981::aid-immu1981&#62;3.0.co;2-x. [DOI] [PubMed] [Google Scholar]
  • 41.Akahoshi T, Sasahara T, Namai R, Matsui T, Watabe H, Kitasato H, Inoue M, Kondo H. Production of macrophage inflammatory protein 3alpha (MIP-3alpha) (CCL20) and MIP-3beta (CCL19) by human peripheral blood neutrophils in response to microbial pathogens. Infect Immun. 2003;71:524–526. doi: 10.1128/IAI.71.1.524-526.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Morel C, Badell E, Abadie V, Robledo M, Setterblad N, Gluckman JC, Gicquel B, Boudaly S, Winter N. Mycobacterium bovis BCG-infected neutrophils and dendritic cells cooperate to induce specific T cell responses in humans and mice. Eur J Immunol. 2008;38:437–447. doi: 10.1002/eji.200737905. [DOI] [PubMed] [Google Scholar]
  • 43.Eum SY, Kong JH, Hong MS, Lee YJ, Kim JH, Hwang SH, Cho SN, Via LE, Barry CE., 3rd Neutrophils are the predominant infected phagocytic cells in the airways of patients with active pulmonary TB. Chest. 2010;137:122–128. doi: 10.1378/chest.09-0903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Seiler P, Aichele P, Bandermann S, Hauser AE, Lu B, Gerard NP, Gerard C, Ehlers S, Mollenkopf HJ, Kaufmann SH. Early granuloma formation after aerosol Mycobacterium tuberculosis infection is regulated by neutrophils via CXCR3-signaling chemokines. Eur J Immunol. 2003;33:2676–2686. doi: 10.1002/eji.200323956. [DOI] [PubMed] [Google Scholar]
  • 45.Meijer AH, van der Sar AM, Cunha C, Lamers GE, Laplante MA, Kikuta H, Bitter W, Becker TS, Spaink HP. Identification and real-time imaging of a myc-expressing neutrophil population involved in inflammation and mycobacterial granuloma formation in zebrafish. Dev Comp Immunol. 2008;32:36–49. doi: 10.1016/j.dci.2007.04.003. [DOI] [PubMed] [Google Scholar]
  • 46.Tobin DM, Vary JC, Jr, Ray JP, Walsh GS, Dunstan SJ, Bang ND, Hagge DA, Khadge S, King MC, Hawn TR, Moens CB, Ramakrishnan L. The lta4h locus modulates susceptibility to mycobacterial infection in zebrafish and humans. Cell. 2010;140:717–730. doi: 10.1016/j.cell.2010.02.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Peters NC, Kimblin N, Secundino N, Kamhawi S, Lawyer P, Sacks DL. Vector transmission of leishmania abrogates vaccine-induced protective immunity. PLoS Pathog. 2009;5:e1000484. doi: 10.1371/journal.ppat.1000484. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Tvinnereim AR, Hamilton SE, Harty JT. Neutrophil involvement in cross-priming CD8+ T cell responses to bacterial antigens. J Immunol. 2004;173:1994–2002. doi: 10.4049/jimmunol.173.3.1994. [DOI] [PubMed] [Google Scholar]
  • 49.Ravindran R, Rusch L, Itano A, Jenkins MK, McSorley SJ. CCR6-dependent recruitment of blood phagocytes is necessary for rapid CD4 T cell responses to local bacterial infection. Proc Natl Acad Sci U S A. 2007;104:12075–12080. doi: 10.1073/pnas.0701363104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Hinchey J, Lee S, Jeon BY, Basaraba RJ, Venkataswamy MM, Chen B, Chan J, Braunstein M, Orme IM, Derrick SC, Morris SL, Jacobs WR, Jr, Porcelli SA. Enhanced priming of adaptive immunity by a proapoptotic mutant of Mycobacterium tuberculosis. J Clin Invest. 2007;117:2279–2288. doi: 10.1172/JCI31947. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Velmurugan K, Chen B, Miller JL, Azogue S, Gurses S, Hsu T, Glickman M, Jacobs WR, Jr, Porcelli SA, Briken V. Mycobacterium tuberculosis nuoG is a virulence gene that inhibits apoptosis of infected host cells. PLoS Pathog. 2007;3:e110. doi: 10.1371/journal.ppat.0030110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Miller JL, Velmurugan K, Cowan MJ, Briken V. The type I NADH dehydrogenase of Mycobacterium tuberculosis counters phagosomal NOX2 activity to inhibit TNF-alpha-mediated host cell apoptosis. PLoS Pathog. 2010;6:e1000864. doi: 10.1371/journal.ppat.1000864. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Chen M, Divangahi M, Gan H, Shin DS, Hong S, Lee DM, Serhan CN, Behar SM, Remold HG. Lipid mediators in innate immunity against tuberculosis: opposing roles of PGE2 and LXA4 in the induction of macrophage death. J Exp Med. 2008;205:2791–2801. doi: 10.1084/jem.20080767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Divangahi M, Desjardins D, Nunes-Alves C, Remold HG, Behar SM. Eicosanoid pathways regulate adaptive immunity to Mycobacterium tuberculosis. Nat Immunol. 2010;11:751–758. doi: 10.1038/ni.1904. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES