The spleen and thymus were removed and placed in a Falcon tube with RPMI media and stored on ice until labeling with antibodies for flow cytometric analysis. The spleen and thymus were teased apart and disaggregated on top of a cell strainer in a cell culture dish containing 5 mL of cold media (Fluorescence Activated Cell Sorting wash). The cell suspension was transferred to a 50-mL conical tube, centrifuged 10 minutes, resuspended in 5-mL RBC lysis buffer, and incubated for 3 minutes at room temperature. The cells were then centrifuged and washed twice with 15-mL Fluorescence Activated Cell Sorting wash buffer, centrifuged at 350g for 5 minutes, and were resuspended at 5 × 106/mL.9
The cells were labeled with 5 μL one step staining Mouse Treg FlowTM kit (FoxP3 Alexa Fluor [Molecular Probes, Eugene, OR] 488/CD25PE/CD4 Per CP) with 100 μL of cell suspension per 5-μL antibody for 20 minutes) followed by fixing with FoxP3 fix/perm buffer (Biolegend Cat No. 421401) at room temperature for 20 minutes. The cells were then permeabilized in FoxP3 perm buffer (Biolegend Cat No. 421402) for 20 minutes, centrifuged and washed twice with FoxP3 perm buffer. The cells were then labeled with anti-Helios (22f6) (Biolegend Cat No. 137203 Alexa Fluor 647) for 20 minutes at room temperature, centrifuged, washed twice, resuspended, and analyzed by flow cytometry. Lymphocyte and macrophage populations were defined by forward and sideward scatter, according to cell granularity and size. Mean control Treg counts in spleen of 435 was based on our earlier article with n = 10 mice.9
To test for the presence of airway inflammation, a corollary of airway hyperresponsiveness in asthma, we subject the lungs from mice to tracheostomy under ketamine/xylazine anesthesia and histologic examination after inflation fixation in formalin, followed by Hematoxylin & Eosin staining by a pathologist who is blinded to the identities of the samples.16 All abnormalities are graded 1, 2, 3, or 4, on the basis of the intensity and extent of peribronchiolar and perivascular cellular infiltration by a pathologist blinded to the identities of the samples. The pathologist also graded the severity and location of crystal deposition and determined whether the crystals, if present, were polarizable.
Experimental Design: Analyses (Collection of Data, Statistical Analysis, and Interpretation)
Results are expressed as mean ± SD, with n being the number of animals per group. For statistical analyses, responses are converted to their logarithms (log10) and differences among groups are analyzed using analyses of variance for repeated measurements, followed by the Bonferroni or Tukey multiple comparisons with P < 0.05 considered statistically significant.
We seal off the trachea with Vetglue or SurgiLock and examine in 1 month after dust administration. At 1 month, we will use CO2 euthanasia and then measure Treg from thymus and spleen by using the methods described in the preliminary studies section, with flow cytometry for markers of Treg (CD4+CD25+FOXP3+).
Histologic sections of Hematoxylin & Eosin–stained lung were graded by a blinded pathologist. We also conduct bronchoalveolar lavage and obtain lung tissue for genomics and proteomics analyses. Analysis of variance is used to compare Treg numbers among the four groups of dust-treated animals, compared with untreated controls.
As added evidence of possible airway inflammation, we perform bronchoalveolar lavage in WT mice. The lungs of each mouse are lavaged three times with 1 mL of phosphate-buffered saline, including an EDTA-free protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN). The bronchoalveolar lavage fluid is centrifuged at 400g for 5 min at 21°C, and the supernatant is analyzed by a quantitative enzyme-linked immunosorbent assay (Assaygate) for selected inflammatory cytokines, IL-2, IL-4, Interferon-γ, IL-10 (Table 3).
Particle sizes of 5 μm or less are present in the Iraq dust sample. The particles are angular in shape, with sharp edges, but are not hollow. Titanium, iron, silicon, calcium and other metals, are present in Iraq dust as shown by energy-dispersive x-ray analysis (Fig. 2). Histologic airway inflammation and interstitial inflammation are not present in untreated C57BL/6 mice but are greatest in Iraq dust–exposed mice compared with San Joaquin or Montana dust experimental groups. The Iraq dust–treated group had the most crystals (which were polarizable), septate thickening, and inflammation. The San Joaquin dust generated less inflammation as a negative control compared with Montana dust (laden with titanium) as a positive control. The overall increase in the triad of crystals, septate thickening, and inflammation in the Iraq dust–exposed mice suggests that titanium alone, while deleterious, does not account for the entire pathophysiologic process.
The immunopathogenesis of Iraq dust lung injury supports suppression of activated Treg (CD4+CD25+FOXP3+) cells, which are immune-tolerant lymphocytes. These are anti-inflammatory. Suppression of total Treg (CD4+CD25+) is associated in these experiments with upregulation of proinflammatory cytokine IL-2. In contrast, IL-4, which skews B cells to produce the allergic antibody immunoglobulin E, is not increased. The Montana dust, titanium-laden, strongly upregulated anti-inflammatory cytokine IL-10 uniquely among the groups.
A pathologist blinded to the identities of treated mice scored for the presence of crystals, septate thickening, or inflammation, using a grading scheme for 1 = no crystal, 2 = few crystals, and 3 = numerous crystals; and 1 = no septate thickening versus 2 = septate thickening present. A total score added the presence of all three characteristics. Crystals were identified by birefringence in polarizing light microscopy (Fig. 3).
Three mice exposed to Iraq dust had the highest mean scores or 5.3, versus 5.0 for San Joaquin, 4.5 for Montana, and 3.0 for no dust. The presence of titanium in Montana dust but lower score thresholds suggests that titanium alone is not sufficient to induce the combined presence of numerous crystals, septate thickening, and inflammation.
Regulatory T cells as defined by CD4+CD25+T cell percentages of total cells counted from thymus were lowest among the mice exposed to Iraq dust, suggesting the most immunosuppression. Treg are immune-tolerant cells; loss of Treg suggests lack of immune tolerance. In contrast, Treg were highest in mice exposed to dust from Montana. Even though Treg in Iraq dust–exposed lungs were lowest among the three dust-treated groups, this number was similar to WT mice. Because the thymus is the central origin of Treg, a surrogate may be the spleen, because peripheral immune tolerance occurs in the latter organ.
Splenic Treg were lowest in the Iraq dust–exposed mice versus all other groups, including WT, Montana, and San Joaquin, California dust–exposed groups. These data indicate suppression of immune-tolerant CD4+CD25+ regulatory T cell numbers in the periphery.
Triple-staining CD4+CD25+FOXP3+ Treg indicate activation and survival. Percentages of these CD4+C25+FOXP3+ cells were all lower in dust-treated groups, than that in untreated mice. In descending order of percentages: Montana, followed by Iraq, then San Joaquin, California in thymus. In the spleen, numbers of triple-staining, CD4+CD25+FOXP3+ Treg were lower in Iraq and San Joaquin dust–exposed mice, compared to WT and Montana experimental arms.
Interleukin 4 was nearly undetectable, thus supporting the concept that this exposure does not induce an immunoglobulin E–mediated allergic response. Interferon-γ levels were less pronounced than IL-2 but were highest in Iraq and untreated groups, possibly induced as a counter-regulatory response. Anti-inflammatory IL-10 was lowest in the Iraq group among the dust, with Montana and no dust–exposed groups having the highest response. Interleukin 6 was higher in San Joaquin compared with the other groups, which were negligible.
Histologic examples of lungs from each experimental arm shows no dust at 400× with normal histology in C57BL/6 mouse lung; San Joaquin, California dust with a minimal amount of focal lymphocytic accumulation in the lower right hand corner of the field; Montana dust with significant inflammation, more widespread; and Iraq dust with septate thickening, interstitial inflammation, incompletely phagocytosed crystals. The highest total lung injury score was in the Iraq dust–exposed group, with more crystals, which were polarizable, consistent septate thickening, and inflammation, both airway and interstitial.
Particulate matter air pollution induces vascular inflammation and is associated with premature death from cardiovascular and lung disease, including myocardial infarction and asthma exacerbations.17,18 In the case of particulate matter air pollution from an Iraq dust sample, we now know that dust size is respirable, with 2.5 μm sized particles present, and that these particles exhibit sharp edges. The physical properties are a concern, akin to asbestos fibers, as the histologic slides show incomplete phagocytosis by pulmonary alveolar macrophages. A plausible theory as to shape is that heavy trucks and tanks may have crushed surface dust to alter its rheology. These are solid particles. We did not see hollow particles discussed by Captain Mark Lyles at the First Scientific Symposium on “Lung Health After Deployment to Iraq and Afghanistan,” Stony Brook University, March 2011(personal communication, Captain Mark Lyles, US Naval War College, Newport, Rhode Island). So, these particles cannot transport substances as containers or “nano-carrier vehicles.” Concentrations of trace metals and minerals in the dust are concerning, because some forms of titanium may be profibrotic19 and are not digestible, and calcium is an airway irritant.
Instillation of Iraq dust yields lymphocytic airway inflammation and interstitial changes which are patchy. Subtle changes in lungs of patients with dust inhalation may not necessarily be visualized with chest x-rays or observed with in-office spirometry, as seen in the cohort of patients studied by Matt King and Robert Miller's group.20 These patients have lungs which reflect the concept that histologic bronchiolitis and vascular remodeling may be present in the absence of radiographic and pulmonary function physiologic abnormalities. The polarizable crystals we see in mice with dust inhalation are similar to that reported by Miller's group. Among the three patients we have examined by open lung biopsy, all have crystals, and titanium is the suspected culprit by micro x-ray fluorescence.
Interleukin 2 is a proinflammatory cytokine expressed by lymphocytes, which enhances further lymphocyte proliferation. The striking finding of higher levels of IL-2 in Iraq dust–treated mice supports a proinflammatory milieu, especially because anti-inflammatory IL-10 levels are not upregulated. This is a specific inflammation profile, because IL-6, another proinflammatory cytokine, notably increased in acute respiratory distress syndrome from lung stretch,21 is not increased. Interferon-γ is also not increased.
Regulatory T cells are immune-tolerant cells that are anti-inflammatory. They are centrally located in the thymus and also may be found in the periphery from a splenic origin. Finding suppressed Treg staining for CD4+CD25+ surface markers suggests that there is peripheral immunosuppression in dust-treated mice.
Although a plethora of airborne hazards may have been experienced by military personnel during deployment to Iraq—detonation of mortars by Al-Qaeda, improvised explosive devices, vehicle improvised explosive devices, blast overpressure from shock waves resulting from explosions, dust and sandstorms called Shamal and Sharq, fumes from burning trash with jet fuel Jet Propellant Eight in continuously incendiary burn pits, indoor and outdoor aeroallergens (depending on geographic location), infectious vectors from organic components in the dust and air—both bacterial and viral and fungal—our model allows the isolation of dust and the ability to examine its sundry inorganic components and their effect on lung health. Because only half of patients with new-onset dyspnea after deployment to Iraq and Afghanistan in our cohort1 respond to asthma medications, targeting the specific, characteristic immune response with respect to Treg and IL-2 may be a more-focused approach.
Although this research provides some insight into the role of dust encountered in Iraq and Afghanistan wars on the production of respiratory disease observed in some of the troops deployed there, more work is needed to understand the role of other airborne contaminants in producing these effects. In addition, the study results are based on only dust samples obtained at Camp Victory, Iraq. Furthermore, the number of animals tested was relatively small. Holding some animals beyond 1 month to evaluate long-term effects would be of interest.
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