Mycobacterium immunogenum genotypes 700506, MJY-3 MJY-4, MJY-12 and MJY-14, originally isolated from diverse contaminated metal working fluids [7], [5] were maintained by sub culturing on Middlebrook 7H10 agar or in Middlebrook 7H9 broth (Difco Laboratories, Sparks, MD, USA) and storing as frozen glycerol stocks in the same medium. Each genotype was grown to mid-log phase in Middlebrook 7H9 broth (Difco Laboratories, Sparks, MD, USA) supplemented with 10% Oleic acid-Albumin-Dextrose-Catalase (OADC) enrichment medium (BD Biosciences, Sparks, MD, USA) and 0.5% glycerol with continuous shaking (150 rpm) at 37°C to a 150 Klett reading (equivalent to approximately 10⁹ cfu/ml) measured by using a Klett photoelectric colorimeter (Klett, New York, NY, USA). Shaking reduced the cell clumping thereby facilitating the subsequent procedure for generation of a monodispersed cell suspension (see details below).

Murine alveolar macrophage cell line MH-S (CRL-2019), purchased from the American Type Culture Collection (ATCC), Manassas, VA, USA, and was used. The MH-S cells were maintained in RPMI 1640 medium (ATCC, Manassas, VA, USA) supplemented with 10% fetal bovine serum and 1% streptomycin-penicillin-glutamate solution (Invitrogen, Carlsbad, CA, USA). Cells were grown for 48h at 37°C in a humidified 5% CO₂ incubator, and were collected, counted, and adjusted to a concentration of 1×10⁶ cells/ml for further use. The alveolar macrophage cells were seeded in 12-well culture clusters at a density of 1×10⁶ cells/well 12 hours prior to the bacterial challenge.

Preparation of single-cell suspensions of M. immunogenum genotypes for infection For all macrophage infection experiments with M. immunogenum 700506, single cell suspension for use as inoculum was prepared as follows. A liquid culture (50 ml) grown to mid-log phase (150 Klett reading) was centrifuged at 3000 x g for 15 min and the cell pellet resuspended in 10 ml of complete RPMI medium (without antibiotics). The suspension was passed serially (ten times) through a 20 gauze syringe needle using a glass syringe and then through a 25 gauze syringe needle. Remaining small clumps were removed with additional centrifugation at 350 × g for 5 min. The resulting single cell suspension, confirmed based on microscopy, was quantified (colony forming units/ml) by spread plate method using Middlebrook 7H10 agar (supplemented with OADC). A freshly prepared inoculum prepared using this optimized protocol was used for macrophage infections. For genotype comparison experiments, individual genotypes were first grown on 7H10 agar-OADC plates and 10 loopfuls of bacterial cells from each plate were separately resuspended in 10 ml of complete RPMI medium and single cell suspensions were prepared by serial passaging through syringe needles as described above. All inocula were normalized to 10⁸ cfu/ml before use in infection experiments.

M. immunogenum genotype 700506 was used as a reference strain to investigate the effective dose- and time- of exposure. The selected dose-time combination was then used for comparing the different genotypes. The freshly propagated MH-S cells, first allowed to adhere as monolayers in 12-well culture clusters (1×10⁶ cells/well), were infected with the 700506 inoculum at varying multiplicity of infection (MOI), ranging from .001 (10³ cfu/10⁶ MH-S cells) to 1000 (10⁹ cfu/10⁶ MH-S cells) MOI. MH-S cells stimulated with lipopolysaccharide (LPS, Sigma Aldrich, St. Louis, MO) @ 500 ng/ml served as positive control for assay validations. The MH-S cells treated with an equal volume of the vehicle (antibiotic-free RPMI medium) were used as negative control. Each treatment was carried out in triplicate. Subsequently, the experimental 12-well cultures challenged with the selected effective infection dose were incubated at 37°C in a humidified 5% CO₂ incubator for varying periods of time (3h, 6h, 12h and 24h). At each time point, the treated cells were harvested and subjected to further analyses. The selected effective pathogen dose and time combination was then used to compare the other four genotypes (MJY-3, MJY-4, MJY-12 and MJY-14).

In an independent experiment, the MH-S cells were treated with MAP kinase inhibitors, using two different doses (20 µM and 40 µM) of the p38 inhibitor SB202190 (EMD Biosciences, San Diego, CA), or the JNK inhibitor SP600125 (EMD Biosciences, San Diego, CA), 1h prior to the challenge with M. immunogenum 700506. Positive (no-inhibitor) and negative (vehicle) controls were run in parallel for comparison. Macrophage cells were harvested at 24h post-exposure and subjected to further analysis, as described below.

Cytotoxicity in the pathogen-challenged MH-S cells was measured in terms of release of lactate dehydrogenase (LDH) using Cyto Tox96 non-radioactive cytotoxicity assay kit (Promega, Madison, WI, USA) per manufacturer’s instructions.

Macrophage cell viability changes were estimated using trypan blue staining (Invitrogen, Carlsbad, CA, USA) for microscopic counting of the live and dead cells using hemocytometer (Bausch & Lomb Rochester, NY USA).

The levels of NO in the culture supernatants were estimated as nitrite using the Griess reagent system per manufacturers’ instructions (Promega, Madison, WI, USA).

Levels of different cytokines (TNF-α, IL-6, 1L-1α, IL-1β, and IL-10 and GM-CSF) were measured in the culture supernatants or cell lysates from macrophage monolayers infected with M. immunogenum genotypes. Sandwich ELISA kits were used to detect the cytokine levels, following the manufacturer’s protocol and instructions (eBioscience, San Diego, CA, USA).

MH-S cells (1×10⁶ cells/well) were adhered for 4 hours followed by washing (three times) with RPMI medium-without-antibiotics (RPMI-A). The cells were infected with MI 700506 at 10 multiplicity of infection (MOI) for 1hour, followed by washing (3x) with RPMI-A medium and incubation in presence of gentamycin (10 µg/ml) for 30 min. to kill extracellular bacteria in the medium. The infected cells were again washed (3x) followed by incubation in RPMI-A medium. Efficacy of the gentamycin treatment in terms of complete inactivation of the extracellular bacteria was confirmed by plating the culture supernatant on Sauton’s agar. For determination of intracellular pathogen load, the cells were lysed at different time points of incubation, using SDS (0.25%) and neutralized using bovine albumin (0.1%). Serial dilutions of the lysate were spread plated on Sauton’s agar for determination of the intracellular pathogen load in terms of colony forming units (CFUs).

MI 700506-infected (24 hours) and uninfected (control) MH-S cells were washed twice in 0.175 M sodium cacodylate buffer (pH 7.4) and fixed using 3% gluteraldehyde. The fixed cells were processed for transmission electron microscopy at the Cincinnati Children’s Hospital Medical Center Pathology Research Core facility, using their standard procedures. Images were taken at a magnification of 30,000 X using AMT digital camera system (Advanced Microscopy Techniques, Corp., Woburn, MA, USA).

MH-S cells (1×10⁶/well) adhered for 4 hours in a 12-well plate were infected with MI 700506 for 60 min. using various MOIs, ranging from 0.001 to 1000. The infected cells were washed (3x) with RPMI-A and lysed with RIPA buffer containing a protease and phosphatase inhibitors cocktail. Western blot analysis was performed using the primary antibodies anti-phospho p38, anti-p38, anti-phospho SAPK/JNK, and anti-SAPK/JNK (all from Cell Signaling Technology, Boston, MA, USA), each at a dilution of 1∶1000. Anti-β Actin was used at a dilution of 1∶10,000 (Sigma, USA). HRP-conjugated anti-rabbit and anti-mouse secondary antibodies (Cell Signaling Technology, Boston, MA, USA) were used at 1∶1000 and 1∶2000 dilutions, respectively. The bands were visualized with an ECL kit per manufacturer’s instructions (Pierce Chemical, Rockford, IL, USA) and subjected to densitometric quantification using the NIH Image J software.

The data are presented as means ± standard deviation (SD) obtained from at least three independent experiments, each performed in triplicate. Differences between groups were assessed by the paired two-tailed Student’s t test with level of significance P≤0.05 (n = 9) being accepted as statistically significant.

Alveolar macrophages infected with MI 700506 showed dose- and time-dependent immunological and cellular responses.

Macrophage cytotoxicity and cell death. LDH release, an indirect indicator of cytotoxicity, due to the loss of cell membrane integrity or cell lysis, increased in a dose- and time- dependent manner in the MH-S cells infected with MI 700506. The LDH release (Fig 1A) began at 0.001 MOI and continued to show a steady increase with dose through 100 MOI. This was followed by a more dramatic increase in the next higher dose (1000 MOI). The more direct assay for viability loss (Trypan blue exclusion-based assay) yielded a dose-response pattern quite parallel to the one followed by the LDH release (Fig 1A). Time-course exposure (3h to 24h) of the MH-S cells with 100 MOI (a dose that gave a reasonably high measurable response) showed a significant induction of cytotoxicity as early as 3h which continued through 24h post-infection (Fig 1B). In terms of cell viability, the selected dose caused a slight loss at 3h post-infection but a significant loss after 6h of infection, which continued to increase through 24h post-infection (Fig 1B). This showed that onset of LDH increase preceded the onset of viability loss in the infected macrophages.

Intracellular replication of the pathogen. The fate of M. immunogenum inside the macrophages is not known. To examine whether MI survives and replicates inside the alveolar macrophages post-phagocytosis (1 hour), we followed infection dynamics for 72 hours. The test strain showed a steady increase in intracellular multiplication leading to a 2.6 log increase in pathogen load (Fig 2A). The measured bacterial load was a result of the replicating intracellular bacteria, as gentamycin was shown to completely inactivate all extracellular bacteria (Fig 2B). Direct evidence for the intracellular presence of MI was obtained by Transmission electron microscopy on 24h-infected cells wherein MI was seen localized mostly in vacuoles (Fig 2C and D). We also observed indication of the presence of intracellular bacteria in the macrophage cells by acid fast staining method (data not shown).

NO Production. Extracellular NO production increased in a dose- and time- dependent manner compared to the vehicle control (3.42±0.104 µM). In the dose-response experiment (Fig 3A), there was a significant (p≤0.05) increase in NO production at doses 0.1 MOI (6.04±0.105 µM) through 1000 MOI (12.78±1.25 µM). The increase was more pronounced and comparable at the high doses (100 and 1000 MOI). In the time-course analysis (Fig 3B), the 100 MOI dose resulted in a significant increase in NO induction beginning at the 12h time-point and continuing through 24h post-infection. Unlike cytotoxicity/cell vitality loss, no significant induction was observed at 3h and 6h post-infection, as compared to the vehicle control.

Expression of cytokines. The proinflammatory cytokines TNFα, IL-1β, IL-1α, and IL-6 and GM-CSF were found to be upregulated in a dose- and time- dependent manner whereas the anti-inflammatory cytokine IL-10 was not detected within or outside the cells in the infected macrophage cultures (Fig 4). Similar to the cellular responses, all upregulated proinflammatory cytokines showed an increase in expression with the increase in pathogen dose and the effect was significant at 100 and 1000 MOI (Fig 4A subpanels a through e). In terms of the production kinetics, majority of the inflammatory cytokines were detected as early as 3h post-infection and increased gradually with time reaching a maximum at the highest time point (24h) post-infection (Fig 4B- subpanels a through e). Interestingly, IL-1β and IL-1α were detected primarily in cell lysates, indicating their intracellular localization (Fig 4B- subpanels a and b). Significant induction (p≤0.0012) in IL-1α production was also detected extracellularly but only at 24h post-infection at the highest doses (100 and 1000 MOI).

The preceding MI 700506 experiments allowed us to select an effective and realistic dose-time combination (100 MOI for 24h) based on induction of cellular and immunological responses in MHS cells. Using this dose (100 MOI) and exposure time (24h) combination, both inflammatory and cellular damage responses were compared for the five different MI genotypes, namely 700506, MJY-3, MJY-4, MYJ-12 and MJY-14.

Of these, the 700506 genotype gave the highest cellular response. MJY-3 increased LDH release (up to 53.4±5.66%) to significantly (p≤0.0057) higher levels as compared to the other genotypes next only to the 700506 genotype (74.8±13.4%). This cytotoxicity trend paralleled with the observed cell viability loss in the MJY-3 treated group (42.7±12.84 percent) as compared to 700506 (53.63±10.30%) and other genotypes (Fig 5). These results demonstrate that MJY-3 is the most potent among the other four genotypes in terms of inducing a cellular response in alveolar macrophages.

The individual genotypes showed significant but differential response in terms of induction of proinflammatory mediators (cytokines/NO) in the MH-S cells (Fig 6 A-F). In terms of cytokine expression, MJY-3, MJY-4, and MJY-14 showed relatively higher responses than 700506 whereas MJY-12 showed the lowest response. For NO induction, MJY-3 induced the highest amount of NO (6.4±0.5 µM), next only to 700506, as compared to the other genotypes (MJY-14, MJY-4, and MJY-12). Collectively, the results imply that MJY-3 and 700506 are the most potent genotypes for inducing inflammatory and cellular damage responses in alveolar macrophages.

Considering the highest potential of MI 700506 for induction of cellular and immunological responses in MH-S cells, we chose this genotype to examine the role of MAPK pathway signaling in cytotoxicity/cell death and inflammatory response (cytokine/NO expression). Infection of MH-S cells with increasing pathogen dose (0.001 through 1000 MOI) for 1 hour upregulated the total p38 and JNK levels and efficiently phosphoactivated JNK and p38 in a dose-dependent manner, as evident from the immunoblots obtained using total- and phospho- p38 (Thr180/Tyr182) and total- and phospho-JNK( Thr183/Tyr185) specific antibodies (Fig 7A and B). Unlike JNK expression levels which were upregulated significantly at all infection doses, p38 levels required a minimum infection dose of 0.1 MOI for its upregulation. The dose-response for JNK upregulation however showed a non-linear trend unlike p38 which showed an almost linear dose-dependent trend (Fig 7A). The phosphoactivation did not follow the same trend as the upregulation (Fig 7B). For instance, p38 was phosphoactivated at all doses whereas JNK was activated beginning 0.01 MOI. A noticeable difference in p38 and JNK was the differential effect of the highest infection dose (1000 MOI); while p38 showed almost similar effect of 100 and 1000 MOI, JNK showed a decrease in levels and activation at the highest dose (1000 MOI).

Alternately, the MH-S cells were pretreated with p38 and JNK inhibitors to understand the role of MAPKs based on inhibition. Interestingly, use of a p38 inhibitor (SB202190) or a JNK inhibitor (SP600125) 1h prior to challenging with 700506 led up to > 80% (p≤0.0051) decrease in the tested cytokines including IL-1α, IL-1β, TNF-α, IL-6, and GM- CSF (Fig 8A to E) as well as a significant inhibition of NO induction (Fig 8F). On the other hand, the two inhibitors showed a somewhat opposite effect on cellular damage response (Fig 9A); the effect was further confirmed using increasing doses of the inhibitors (Fig 9B). Taken together, the MAPK inhibition (p38 and JNK) abrogated in part the proinflammatory response suggesting the role of MAPK pathway in HP immunopathogenesis.