The IL-23/Th17 axis is involved in the adaptive immune response to Bacillus anthracis in humans

hDCs engulf and kill BA

To study their ability to phagocytose and kill BA spores, we challenged hDCs with germination-proficient or germination-deficient (Δgerh) BA spores in vitro and enumerated the number of viable intracellular bacteria within the hDCs over time. As seen by electron microscopy, BA spores were visible within hDCs as early as 30 min postinfection at a multiplicity of infection (MOI) of 1:1 (Fig. 1A). The uptake of BA spores by hDCs was not uniform and some hDCs had multiple spores (Fig. 1A(i)), whereas others had two spores (Fig. 1A(ii)) or no spores at all (data not shown).

Murine macrophages kill BA when infected with germination-proficient BA spores, but not mutant spores that are defective in their ability to germinate 3. Therefore, we examined the capacity of hDCs to kill BA by infecting them with germination-proficient BA spores and BA spores (Δgerh), the latter with a deletion in the germination operon that results in delayed germination. We found that at early time points postinfection (16 h), hDCs efficiently killed BA that had germinated into vegetative bacilli, but were incapable of killing Δgerh spores (Fig. 1B). Notably, nongerminated spores could be recovered from viable hDCs up to 48 h following infection with Δgerh spores as evidenced by their resistance to heat (65°C for 30–45 min). In contrast, infection with increasing doses of germination-proficient BA spores resulted in a marked decrease in the viability of hDCs (data not shown). This suggests that hDC viability is adversely affected when increasing numbers of intracellular BA spores germinate into viable BA bacilli.

BA infection elicits Th17- and Th1-promoting cytokines from human moDCs

Upon encountering microbes or microbial products, DCs produce cytokines and chemokines, and upregulate surface expression of HLA and costimulatory molecules that direct the adaptive immune response. The cytokine profile of responding DCs largely dictates the type of Th-cell response that will ensue. Thus, it is paramount that DCs exposed to bacterial pathogens, such as BA, produce proinflammatory cytokines that drive powerful Th17 and Th1 responses considered essential for host protection.

Increasing evidence identifies IL-1β, IL-6, and IL-23 as key mediators in the development and maintenance of human Th17 responses 4-8. This relatively newly described Th-cell subset, which produces IL-17 cytokines as well as IL-22 and/or IFN-γ, has been implicated in a number of inflammatory diseases 8-10, but is also considered critical for protection against bacterial and fungal pathogens 11-15. Given the importance of IL-23 in Th17-cell activation and host protection from bacterial infections 16, 17, we looked to see if BA spores stimulate hDCs to produce IL-23 and other proinflammatory mediators reported by Pickering et al. 18. hDCs from healthy donors were infected with increasing doses of germinating BA spores or mutated Δgerh spores and culture fluids analyzed for IL-23, as well as IL-1β, IL-6, and IL-12 proteins the following day. We found that BA spores triggered hDCs to secrete IL-23 in a dose-dependent manner (Fig. 2A). Consistent with previous reports, IL-1β, IL-6, and IL-12 were also increased in culture fluids from hDCs infected with both germination-proficient and -deficient BA spores (Fig. 2B), and DC activation and maturation markers were upregulated on the surface of BA-infected DCs (Fig. 2D) 18-20. The percent of CD80- and CD83-expressing hDCs were increased, whereas the densities of HLA-DR and CD40 molecules were augmented on the surface of hDCs infected with BA.

Somewhat surprisingly, germinating BA spores and mutated Δgerh spores stimulated hDCs to secrete similar amounts of IL-1β and IL-23, whereas germinating BA spores induced superior production of IL-6 and IL-12 (Fig. 2B). These intriguing results suggest that two different mechanisms trigger production of IL-1β/IL-23 and IL-6/IL-12 in hDCs, which has also been reported for hDCs infected with the intracellular gram-negative bacteria, Chlamydia trachomatis 21. Given that IL-12 and IL-23 responses to agonists of PRRs are regulated differently in monocyte-derived hDCs 22-25, one could speculate that mutated Δgerh spores lack additional signaling components found in germinating spores that augment IL-6 and IL-12 responses. It is also possible that the spore form induces IL-1β and IL-23, but that only the bacillary form induces IL-6 and IL-12.

We were the first to show that IL-1β controls IL-23 production in human monocytes 26. Since germination-proficient BA as well as Δgerh spores stimulated hDCs to produce similar levels of these two cytokines (Fig. 2B), we predicted that the IL-23 response of hDCs to BA spores was regulated by IL-1 receptor mediated signaling. Addition of the naturally occurring IL-1 receptor antagonist (IL-1ra) to hDCs infected with BA spores, significantly inhibited IL-23 production (Fig. 2C), indicating that the IL-23 response of hDCs to BA spores is, at least partly, mediated by IL-1 receptor signaling. Thus, IL-1β may play a critical role in the immune response to BA in humans at both the level of DC activation, i.e. autocrine cytokine production and Th17-cell differentiation.

hDCs loaded with germinating BA spores trigger contact-dependent and antigen-specific Th17 responses

The Th17 pathway contributes to host protection against extracellular bacterial and fungal infections 27. As such, patients with defects in genes involved in Th17 development suffer from recurrent extracellular microbial infections 28, 29. The cytokines IL-1β, IL-6, and IL-23 are important mediators of Th17 (and memory Th1) responses 4, 5, 8, 9, 30, whereas IL-12 drives differentiation of Th1 cells 22, 31. Since hDCs infected with germinating BA spores produced all of these cytokines, we tested the hypothesis that BA-infected hDCs stimulate BA-specific Th17 and Th1-cell responses.

Highly pure CD4⁺ T cells (>92%) were obtained from PBMCs of healthy individuals using a negative selection kit from Stemcell Technologies (Fig. 3A). Th17 cells, which express IL-1RI, IL-23R, and CCR6 could be identified in a portion of circulating CD4⁺ T cells isolated from all healthy individuals tested (Fig. 3A). CD4⁺ T cells were then cultured with autologous hDCs infected with no spores, Δgerh or BA spores in the presence of recombinant human IL-7 and IL-15 for 7 days to facilitate induction of a primary T-cell response. Semipermeable transwell inserts were used to prevent CD4⁺ T cells from contacting BA-infected hDCs, as a means of assessing contact-dependent versus contact-independent, cytokine-induced T-cell activation. At the end of the week long co-culture, cell-free supernatants were collected and analyzed for secreted IL-17A and IFN-γ proteins, the hallmark cytokines of Th17 and Th1 cells, respectively.

As anticipated, hDCs infected with BA spores stimulated IL-17A and considerable quantities of IFN-γ in co-cultures with autologous CD4⁺ T cells (Fig. 3B). Importantly, IL-17A and IFN-γ responses were significantly inhibited when cellular contact between CD4⁺ T cells and BA-infected hDCs was prevented using 0.4 μm semipermeable transwell inserts (Fig. 3B). These results provide primary evidence that BA-infected hDCs induce IL-17A and IFN-γ, suggesting that the responses are likely contact dependent and not merely the result of soluble mediators released by BA-infected hDCs.

Although IL-17A and IFN-γ responses tended to be augmented in co-cultures containing hDCs infected with germinating BA spores compared with those of co-cultures with hDCs infected with germination-deficient Δgerh spores, the differences were not consistent for all individuals tested. This finding is somewhat unexpected, given the disparate production of IL-6 and IL-12 we observed in hDCs infected with BA spores compared with that of Δgerh mutants (Fig. 2B). However, it is highly possible that during the week long co-culture necessary for T-cell priming, the mutated Δgerh spores germinated, thereby negating the distinct cytokines responses we detected the day following infection with germinating BA spores or Δgerh mutants (Fig. 2B). Nevertheless, these findings indicate that both IL-17-producing Th17 and IFN-γ-producing Th1 cells respond to BA in humans, and in addition, raise a series of questions regarding the cell subset, specificity, and recall potential of the CD4⁺ T-cells responding to BA-infected hDCs.

To address these central issues, we conducted the following experiments. Cells from the primary studies described above that were primed with hDCs infected with no spores, Δgerh mutants, or BA spores, were harvested, washed to remove cytokines, counted and replated in fresh culture media for an additional 2 days with a second preparation of autologous hDCs infected with no spores, Δgerh mutants, germinating BA spores, or BA antigens LF and PA. Secreted IL-17A and IFN-γ proteins were quantified in the culture supernatants by ELISAs and the cell source of these cytokines identified by intracellular cytokine staining and flow cytometry.

T cells primed (1^ο challenge) and restimulated (2^ο challenge) with BA-infected hDCs elicited nearly twofold greater IL-17A and IFN-γ responses compared with (i) T cells primed with BA-infected hDCs and restimulated with uninfected, Δgerh-, LF- or PA-infected hDCs, (ii) T cells primed in the absence of spores and restimulated with BA-infected hDCs, or (iii) T cells primed with Δgerh-infected hDCs and restimulated with BA-infected hDCs (Fig. 4A). Notably, hDCs secondarily loaded with (i) mutant Δgerh spores, (ii) LF, or (iii) PA were incapable of recapitulating the secondary IL-17A and IFN-γ responses elicited by hDCs infected with germination-proficient BA spores (Fig. 4A). In addition, the neo-antigen keyhole limpet hemocyanin (KLH) was not effective at restimulating T cells primed with BA-infected hDCs and vice versa (Fig. 4B, left and right panels). Taken together, these results strongly suggest that optimal BA-specific, proinflammatory CD4⁺ T-cell responses are elicited by BA spores that germinate into replicating bacilli, and the immunogenic epitope(s) are distinct from LF and PA antigens previously described by others 32-34.

IL-17A and IFN-γ can be produced by separate CD4⁺ T cells or within the same CD4⁺ T-cell. Consequently, cells that produce IL-17 alone or together with IFN-γ are recognized as Th17 cells, whereas sole producers of IFN-γ are generally considered Th1 cells 35, 36. Since both of these antimicrobial cytokines were produced in a BA-specific manner, we wanted to determine whether distinct or overlapping populations were the cell source in our in vitro system. Intracellular cytokine staining was performed on T cells primed and restimulated with hDCs infected with germinating BA spores, while T cells primed with uninfected hDCs (no spores) and restimulated with BA-infected spores served as a control. To optimize intracellular cytokine detection, PMA and ionomycin were added to both conditions in the presence of monensin for the last 5 h of the 2 day restimulation period, and the cells analyzed by multiparameter flow cytometry.

CD4⁺ T-cell populations producing IL-17A alone, IFN-γ alone, or these two cytokines simultaneously comprised both culture conditions. However, the proportion of CD4⁺ T cells producing both IL-17A and IFN-γ (double producers) increased more than twofold in T-cell cultures primed and restimulated with BA-infected hDCs compared with that of T cells primed with uninfected hDCs and restimulated with BA-infected hDCs (Fig. 4C). These studies demonstrate, for the first time, that hDCs infected with germinating BA spores activate Th cells that simultaneously produce IL-17A and IFN-γ, a profile indicative of pathogen-induced Th17 cells 35-37.

BA spores and antigens

The BA Sterne strain, 34F2, was originally obtained from Dr. Les Baillie (Cardiff, Wales), and the germination-deficient Δgerh strain of Sterne 34F2 was obtained from the laboratory of Dr. Phillip Hanna (Ann Arbor, MI, USA). BA PA and LF were purchased from List Biological Laboratories, Inc. (Campbell, CA, USA).

Cell isolation

PBMCs were obtained from the peripheral blood of healthy human donors under a protocol approved by the University of Maryland IRB. PBMC were isolated by density gradient centrifugation in Ficoll-Hypaque according to manufacturer's instructions. Highly pure (>90%) lymphocytes and monocytes were obtained from healthy donors as above followed by countercurrent centrifugation elutriation. Cells were viably frozen using an automated step-down freezer, and stored in liquid nitrogen until used. Upon thawing, cell viability was determined by trypan blue exclusion. Untouched CD4⁺ T cells (>95% pure) were purified from lymphocytes by magnetic cell separation using the EasySep Negative Selection Human CD4⁺ T-cell Kit (Stemcell Technologies). For some experiments, monocytes were isolated from thawed PBMC by magnetic cell separation using the EasySep Negative Selection Human Monocytes without CD16 Depletion Kit (Stemcell Technologies).

Generation and infection of hDCs with BA spores

To generate immature, monocyte-derived hDCs, human monocytes were cultured incomplete RPMI (RPMI-1640, 10% FBS, 1% l-glutamine, 1% Pen-Strep, and 20 mM Hepes) (Invitrogen) supplemented with 50 ng/mL GM-CSF plus 25 ng/mL IL-4 (both from R&D Systems) at 10⁶ cells/mL for 3 days. hDCs were collected, washed, counted, and phenotyped for DC markers CD1a, CD11c, CD40, CD80, CD83, CD86, and HLA-DR by flow cytometry. To infect hDCs, the cells were washed and resuspended in serum-free X-Vivo 10 media (Lonza, MD, USA) without any antibiotics and put into sterile 5 mL polystyrene tubes at 10⁶ cells/mL per tube. BA spores (WT or Δgerh) were added to hDCs at different MOIs (BA:hDC::1:1, 0.5:1, 0.1:1) and incubated at 37°C with 5% CO₂ for 30 min. After 30 min, 50 μg/mL of gentamicin was added to hDCs and incubated for an additional 30 min to kill any extracellular vegetative bacilli. hDCs were washed twice with 1 mL of X-vivo 10 media to remove the antibiotic and any uninternalized spores. In some experiments, live or heat-killed BA bacilli were added to hDCs at an MOI of 1:1. hDCs were then used as described in the following functional assays.

hDC killing of WT and Δgerh BA spores

Following the second wash, hDCs were lysed with 1 mL of sterile distilled water to release the intracellular bacteria. Viable bacteria were enumerated by plating samples on LB-agar plates and counting the number of colonies formed at time 0. For the other time points (6 and 20 h), hDCs were incubated in X-vivo 10 with gentamicin throughout the incubation period, washed twice with 1 mL of X-vivo 10 media to remove the antibiotic, lysed with 1 mL of sterile distilled water, and plated on LB-agar plates. The killing was calculated by subtracting the number of bacteria at different time points from the initial number at time zero, generating a Δlog kill value. As a negative control for killing, hDCs were infected with Δgerh BA spores, which do not germinate for extended periods of time, and therefore are not killed 2.

Cellular responses of hDC to BA spores

hDCs infected with and without BA spores (WT or Δgerh) were resuspended at 10⁶ cells/mL in X-Vivo 10 with gentamicin, and incubated at 37°C with 5% CO₂ for 20 h. Cellular responses were determined by secretion of proinflammatory cytokines by ELISAs and increased expression of activation and maturation markers on the surface of hDCs by flow cytometric analysis.

Primary and secondary T-cell responses to BA

The in vitro human T-cell assay was adapted from standard assays conducted for the study of human immune responses 54. Briefly, hDCs from healthy human donors were infected with BA spores (WT or Δgerh) at a MOI of 1:1, or were left untreated. A total of 10⁵ control or infected hDCs were then cultured with 10⁶ autologous CD4⁺ T cells per milliliter in cRPMI supplemented with 5 ng/mL IL-7 and 5 ng/mL IL-15 (both from R&D Systems) in 24-well plates for 7 days (primary). In some experiments, 0.4 μm semipermeable transwell inserts (Sigma) were used to prevent cellular contact between CD4⁺ T cells and BA-loaded hDCs. To assess secondary recall T-cell responses to BA, cells from the 7-day co-culture were harvested, washed, counted, and replated at a hDC:TC ratio of 1:10 in 24-well plates for 48 h with a second preparation of autologous hDCs that were infected with BA spores (WT or Δgerh) at a MOI of 1:1, 2.5 μg/mL PA, 1 μg/mL LF, KLH (10 μg/mL), or left untreated.

Cytokine ELISAs

Cell-free culture fluids from control and BA-infected hDCs were analyzed for secreted IL-1β, IL-6, IL-12p70, and IL-23 hetero-dimer by ELISAs (eBioscience) following the manufacturer's protocols. Supernatants from primary and secondary exposure of Th cells to uninfected and infected hDCs were analyzed for IL-17A and IFN-γ by ELISAs (eBioscience) following the manufacturer's protocols.

IFN-γ ELISpot

hDCs were infected at a MOI 1:1 with BA spores or mock-infected, then washed and co-cultured with autologous lymphocytes for 7 days (primary) as described above. Cells from the 7 day co-culture were harvested, washed, counted, and replated at 10⁴ cells/100 μL per well with no spores, BA spores, or KLH in 96-well ELISpot plates (Millipore) coated with anti-human IFN-γ (Mabtech) capture antibody. Cell cultures were incubated at 37°C in 5% CO₂ for 48 h and developed per manufacturer's instructions (Mabtech). Plates were stored overnight in the dark at room temperature, and spots were counted using a VersaScan microplate reader (Velocity 11, Palo Alto, CA, USA). The mean totals of IFN-γ spot-forming cells in triplicate wells were determined and expressed as numbers of spot-forming cells per 10⁴ lymphocytes.

Surface and intracellular flow cytometric analysis

Surface expression of CD1a, CD11c, CD14, CD40, CD80, CD83, CD86, and HLA-DR (all conjugated mAbs from BD Pharmingen) was monitored on hDCs pre- and post-BA infection by flow cytometry. The purity of CD4⁺ T cells and proportion of CD4⁺ T cells expressing CD45RO (BD Pharmingen), CCR6, IL-1RI, and IL-23R (all from R & D Systems) at the cell surface was also determined by flow cytometry. A total of 10 000 live cells were acquired on a Canto flow cytometer (BD Pharmingen) and analyzed using FlowJo software and appropriate conjugated immunoglobulin isotype controls (BD Pharmingen).

To detect intracellular cytokines produced by CD4⁺ T cells re-exposed to BA-infected hDCs, 50 ng/mL PMA and 750 ng/mL ionomycin were added to hDC:TC co-cultures for the last 6 h of the 48-h rechallenge. For the last 4 h, 10 μg/mL monensin was also added to these co-cultures. Cells were labeled with anti-human CD3 and CD4 mAbs for detection of surface antigens, then fixed and permeabilized with BD Cytofix/Cytoperm solution and labeled with anti-IFN-γ, anti-IL-17A mAbs (both from eBioscience), or appropriate immunoglobulin isotype controls in BD Perm/Wash Buffer following the manufacturer's instructions. A total of 200 000 CD3⁺ cells were acquired on a BD Canto flow cytometer and analyzed by FlowJo software.

Statistical analyses

Results are displayed as individual data and means ± SD. Statistical significance was determined using two-tailed paired Student's t-test. p values < 0.05 were considered statistically significant.