H. capsulatum G217B [ATCC 26032] is a North American isolate of RFLP class 2 which was termed 'high level' in thermotolerance and pathogenicity. The fungus was grown in Histoplasma-macrophage medium (HMM) broth 36 in a 5% CO₂-95% air atmosphere. Experiments were performed with H. capsulatum grown as yeast cells at 37°C.

Recombinant Yps3p and H proteins was prepared as described previously 3337. For the preparation of crude cell extract, fractionation was done as follows: log-phase yeast cells were pelleted by centrifugation, washed, and resuspended in PBS. They were then disrupted using glass beads in a Mini-Beadbeater-8 (Biospec Products, Bartlesville, OK) at highest setting for three 1 min periods, separated by chilling on ice for 1 min. Beads were removed by low-speed centrifugation and the cell lysate was spun at 15K RPM in a microcentrifuge at 4 C for 30 min. The supernatant was removed as the cytoplasmic fraction. The pellet was resuspended in PBS as the cell wall/membrane fraction.

Microglial cell cultures were purified from wild-type C57BL/6 and TLR2 KO mice (Jackson Laboratories, Bar Harbor, ME) using a method described previously with minor modifications 38. Briefly, cerebral cortical cells from 1-d-old mice were dissociated after a 30 min trypsinization [0.25%] and plated in 75-cm² Falcon culture flask in DMEM (Sigma-Aldrich, St. Louis, MO) containing 10% heat-inactivated FBS (Hyclone Laboratories, Logan, UT)and penicillin/streptomycin (Sigma-Aldrich). The medium was replenished 1 and 4 d after plating. On d 8 of culture, flasks were shaken for 20 min at a speed of 180 rpm in an orbital shaker to remove unattached cells. On d 12 of culture, microglia floating in the media were collected by aspiration, pooled, centrifuged and seeded at appropriate densities after counting. The cells were washed twice with fresh medium 1 h after seeding to remove non-adherent cells. Microglia prepared this way stain 95–98% positive with Mac-1 antibody (Roche Applied Science, Indianapolis, IN).

DNA obtained from the VV Western Reserve strain was used to clone four viral gene products: A46R, A52R, N1L and K1L using PCR. Primers used for amplification were: A46R: Forward: 5'-CAT GCC ATG GCG TTT GAT ATC AGT-3' and Reverse: 5'-CAT GCC ATG GAT GGC GTT TGA TAT-3'; A52R: Forward: 5'-CAT GCC ATG GAC ATA AAG ATA GAT-3' and Reverse: 5'-GTG GAA ATG TCA TAG GCT AGC TAG-3'; N1L: Forward: 5'-CAG GTC ATG AGG ACT CTA CTT ATT-3' and Reverse: 5'-CTA GCT AGC TTA TTT TTC ACC ATA-3'; K1L: Forward: 5'-CAG GAT ATC ATG GAT CTG TCA CGA-3' and Reverse: 5'-CTA GCT AGC TTA GTT TTT CTT TAC AC-3'. PCR was performed on a Gradient 40 Robocycler (Stratagene, La Jolla, CA) using Pfu polymerase (Stratagene) with the following conditions: initial denaturation at 95°C for 2 min 30 sec, followed by 30 cycles of 95°C for 1 min, annealing at 60°C for 1 min and elongation at 72°C for 3 min. Following PCR amplification, viral gene products were purified using a 0.8% agarose gel and were cloned into pORF5-mIL10 (InvivoGen) by replacing the mIL-10 ORF with each VV ORF as described previously 39. This vector carries the murine IL-10 ORF under the control of a composite binary promoter comprised of the elongation factor 1α (EF-1α) and the 5' untranslated region of the human eukaryotic initiation factor 4 g (eIF-4 g). The expression vectors thus generated were termed pORF5-A46R, pORF5-A52R, pORF5-N1L and pORF5-K1L. Expression of these viral proteins was confirmed using Western blot analysis 39.

HEK293T cells, as well as wild-type and TLR2 KO microglia, were transfected with 1 μg pNiFty2-Luc plasmid (InvivoGen) expressing an NF-κB-driven firefly luciferase reporter gene. FuGene 6 was used for transfection of the 293T-mTLR2 cells. Primary microglia are post-mitotic cells which are extremely difficult to transfect using standard methods. In this study, they were successfully transfected using the mouse macrophage nucleofection kit (Amaxa Biosystems, Gaithersburg, MD) and the program Y-01 on the nucleofector I device (Amaxa). Although the transfection efficiency using nucleofection was still low (<10%), luciferase expression occured only in cells that took up the pNiFty2-Luc plasmid. Following nucleofection, the cells were plated in 12-well plates and incubated overnight at 37°C. To stimulate TLR2 signaling, 0.01% heat-killed L. monocytogenes (InvivoGen) was added to the culture medium for 5 h. The cells were then lysed and luciferase activity was measured using Bright-Glo luciferase assay substrate (Promega, Madison, WI) on the IVIS^® Imaging System (Xenogen Corporation, Alameda, CA). Expression levels of the luciferase reported gene were quantified using Living Image^® software (Xenogen). Tranfection efficiencies were tested using a control plasmid expressing green fluorescent protein under the control of CMV IE promoter and the values were normalized to the transfection efficiencies obtained.

A sandwich ELISA-based system was used to quantify CCL2 levels from WT and TLR2 KO murine microglial cell culture supernatants. ELISA plates were coated with rat-anti-mouse CCL2 capture antibodies (R&D Systems, Minneapolis, MN) at 1–2 μg/ml overnight at 4°C. The plates were washed (0.05% Tween-20 in phosphate-buffered saline, PBS) and blocked with 1% BSA in PBS for 1 h at 37°C. Detection antibodies (biotinylated goat anti-mouse CCL2 antibodies, 1–2 μg/ml; R&D Systems) were added for 90 min at room temperature followed by peroxidase conjugated strepavidin (1:3000; Jackson Immunoresearch) for 45 min. A chromogenic substrate (K-blue; Neogen Corporation, Lexington, KY) was then added and color development was stopped with 1 M H₂SO₄. Absorbance values at 450 nm were used to quantify chemokine levels based on the standard concentration curve generated from serial dilutions.

TLRs recognize PAMPs on the surface of pathogens and activate the host's innate immune responses. A number of fungal cell wall components such as mannan, phospholipomannan, and zymosan have previously been shown to be recognized by TLR2 and TLR4 18192940. In order to determine which component of the H. capsulatum cell wall/membrane activated the TLR2 signaling pathway, we have used a stable cell line that expresses murine TLR2 under the control of a composite promoter comprised of the eukaryotic elongation factor-1α (EF-1α) core promoter and the R segment, as well as part of the U5 sequence, of the human T-cell leukemia virus type 1 long terminal repeat 39. These 293T-mTLR2 cells were transfected with the plasmid pNiFty2-Luc, containing the open-reading frame for luciferase under the regulation of five NF-κB binding sites. Using this experimental design, signaling from TLR2 results in the activation of NF-κB which, in turn, activates luciferase expression. Thus, luciferase expression does not occur in the absence of TLR2 signaling. (Fig. 1).

The fungal cell wall fraction was isolated and used to treat 293-mTLR2 cells. Two other fungal proteins Yps3p and H, purified following expression in E. coli, were also tested. H protein serves as a negative control and heat-killed Listeria monocytogenes, a strong inducer of TLR2 signaling, was used as a positive control. As shown in Fig. 2, neither the cell wall fraction (CW) nor the recombinant H protein activated signaling through TLR2. There was no significant luciferase production above the background levels in these samples. On the other hand, the recombinant protein Yps3p induced TLR2 activation that resulted in a marked increase in luciferase production, demonstrating that Yps3p protein is a ligand for TLR2.

To further confirm the role of TLR2 in responding to H. capsulatum, we next attempted to inhibit this signaling pathway using four vaccinia virus([VV) proteins. Among these viral proteins, A46R inhibits signaling from MyD88, the cytoplasmic adaptor of TLR2 41, and A52R interacts with and blocks the activity of two downstream molecules IRAK2 and TRAF6 along the TLR2 pathway 4142; whereas N1L and K1L prevent the release of NF-κB from its inhibitor IκBα 4344 (Fig. 1). ORFs of each of these VV proteins were cloned into the pORF5 vector under the control of a composite binary promoter comprised of the elongation factor 1α (EF-1α) and the eukaryotic initiation factor 4g (eIF-4g). 1 μg of each pORF5-VV plasmid was co-transfected into 293T-mTLR2 cells together with 1 μg of pNiFty2-Luc. Following overnight incubation at 37°C, the cells were treated with Yps3p for 6 h, harvested and the expression levels of luciferase in the transfected cells were measured using a luciferase assay. The data show that NF-κB activation was severely impaired in cells expressing each of these viral proteins when compared to cells expressing pNiFty2-Luc alone, a result which demonstrates that all four viral proteins were able to inhibit Yps3p-induced TLR2 signaling in 293T-mTLR2 cells (Fig. 3). This result not only confirmed Yps3p mediation of TLR2 signaling but also showed that it can be inhibited at different levels (at the receptor as well as downstream) along the TLR2 signaling pathway.

Having determined that H. capsulatum protein Yps3p engages TLR2 signaling pathway and causes NF-κB activation in our 293T-mTLR2 cell line, we went on to determine whether Yps3p protein also induced NF-kB activation in primary brain cells. For this experiment, microglial cells from wild-type C57BL/6 mice as well as TLR2 KO mice were isolated and transfected with pNiFty2-Luc plasmid using nucleofection. After overnight incubation at 37°C, the microglia were exposed to the recombinant fungal protein for 6 h. The cells were then harvested and a luciferase assay was performed. In these experiments, Yps3p-induced TLR2 signaling in wild-type microglial cells resulted in high levels of luciferase expression, demonstrating an increased level of NF-κB activation (Fig. 4). In contrast, a significant reduction in luciferase expression occurred following the identical treatment using TLR2 KO microglia. This result further demonstrates that Yps3p triggered signaling through TLR2.

To further test the role of Yps3p in triggering TLR2 signaling, we performed an ELISA for the proinflammatory immune mediator Chemokine (C-C motif) ligand 2 (CCL2) in wild-type and TLR2 KO microglial cells. The amount to CCL2 secreted into the culture supernatants of microglial cells was quantified using an ELISA assay. While there was no difference in the expression of CCL2 between untreated samples, the expression of CCL2 was elevated in the both wild-type and TLR2 KO microglia following the treatment with Yps3p (Fig. 5). However, the expression levels obtained for TLR2KO microglia were significantly lower than those obtained for wild-type microglia, suggesting that the activation of TLR2 by Yps3p results in the production of CCL2 in these cells.