Structural characterization of gas-phase cysteine and cysteine methyl ester complexes with zinc and cadmium dications by infrared multiple photon dissociation spectroscopy

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Allogeneic Splenocyte Transfer and Lipopolysaccharide Inhalations Induce Differential T Cell Expansion and Lung Injury: A Novel Model of Pulmonary Graft-versus-Host Disease

<div><p>Background</p><p>Pulmonary GVHD (pGVHD) is an important complication of hematopoietic cell transplant (HCT) and is thought to be a consequence of the HCT conditioning regimen, allogeneic donor cells, and posttransplant lung exposures. We have previously demonstrated that serial inhaled lipopolysaccharide (LPS) exposures potentiate the development of pGVHD after murine allogeneic HCT. In the current study we hypothesized that allogeneic lymphocytes and environmental exposures alone, in the absence of a pre-conditioning regimen, would cause features of pGVHD and would lead to a different T cell expansion pattern compared to syngeneic cells.</p><p>Methods</p><p>Recipient Rag1^−/− mice received a transfer of allogeneic (Allo) or syngeneic (Syn) spleen cells. After 1 week of immune reconstitution, mice received 5 daily inhaled LPS exposures and were sacrificed 72 hours after the last LPS exposure. Lung physiology, histology, and protein levels in bronchoalveolar lavage (BAL) were assessed. Lung cells were analyzed by flow cytometry.</p><p>Results</p><p>Both Allo and Syn mice that undergo LPS exposures (AlloLPS and SynLPS) have prominent lymphocytic inflammation in their lungs, resembling pGVHD pathology, not seen in LPS-unexposed or non-transplanted controls. Compared to SynLPS, however, AlloLPS have significantly increased levels of BAL protein and enhancement of airway hyperreactivity, consistent with more severe lung injury. This injury in AlloLPS mice is associated with an increase in CD8 T cells and effector CD4 T cells, as well as a decrease in regulatory to effector CD4 T cell ratio. Additionally, cytokine analysis is consistent with a preferential Th1 differentiation and upregulation of pulmonary CCL5 and granzyme B.</p><p>Conclusions</p><p>Allogeneic lymphocyte transfer into lymphocyte-deficient mice, followed by LPS exposures, causes features of pGVHD and lung injury in the absence of a pre-conditioning HCT regimen. This lung disease associated with an expansion of allogeneic effector T cells provides a novel model to dissect mechanisms of pGVHD independent of conditioning.</p></div

Allogeneic splenocyte transfer followed by inhaled LPS leads to an increase in pulmonary IFN-γ, granzyme B, and CCL5.

<p>Rag1^−/− mice received a transfer of allogeneic (Allo) or syngeneic (Syn) splenocytes followed by daily exposures to aerosolized LPS for 5 days starting 1 week after splenocyte transfer. Mice were euthanized 72 hours after the last LPS exposure and concentrations of proteins were measured in the BAL of AlloLPS and SynLPS mice using multiplex and ELISA assays. (<b>A</b>) IFN- γ is elevated in AlloLPS compared to SynLPS (p = ). (<b>B–D</b>) Th2 cytokines IL-4, IL-5, and IL-13 are similar between AlloLPS and SynLPS. (<b>E</b>) IL-17 is reduced in AlloLPS compared to SynLPS (p = 0.0078). (<b>F</b>) IL-10 is unchanged between AlloLPS and SynLPS. (<b>G</b>) IL-15 is elevated in the BAL of AlloLPS compared to SynLPS (p = 0.014). (<b>H</b>) Granzyme B (Gr B) is elevated in the BAL of AlloLPS mice compared to SynLPS (p = 0.029). (<b>I</b>) CCL5 is elevated in the BAL of AlloLPS mice compared to SynLPS (p = 0.0023). Data represent the average +/− SEM and * = p<0.05 and ** = p<0.005. ND = non detectable. Data have been replicated in 2 independent experiments.</p

After allogeneic lymphocyte transfer followed by inhaled LPS, pulmonary donor-derived cells are comprised primarily of lymphocytes while myeloid cells are of recipient origin.

<p>Rag1^−/− mice received a transfer of allogeneic (Allo) or syngeneic (Syn) splenocytes and underwent daily exposures to aerosolized LPS for 5 days starting 1 week after splenocyte transfer. Mice were euthanized 72 hours after the last LPS exposure and lung cells were analyzed using flow cytometry. For flow analysis, a singlet gate was used to exclude cell aggregates, followed by an all-cell gate to exclude small debris and dead cells, followed by a CD45⁺ cell gate to define all white blood cells (as described in methods). (<b>A</b>) All CD45⁺ cells were separated into donor and recipient-derived cells based on their expression, or lack thereof, of the donor marker CD45.1 (in the case of SynLPS mice on the left) or H2Kk (in the case of AlloLPS mice on the right). The subsequent graphs show flow cytometry plots for AlloLPS lung cells but the same analysis was performed for SynLPS and yielded similar results. (<b>B</b>) Donor- (left) and recipient-derived (right) lung cells were analyzed separately. Representative flow cytometry plots show gating of MHC⁺CD11c⁺ antigen-presenting myeloid cells, which are mainly present in the recipient cells (42.2% vs. 0.9% in donor cells) and are large based on the side by forward scatter graph (far right). The MHCII⁺CD11c⁻ cells are classically comprised of B cells and CD11b⁺MHCII⁺ myeloid cells. MHCII⁻CD11c⁻ cells are usually comprised of neutrophils, monocytes, and T cells. (<b>C</b>) Representative flow cytometry plots show the population of MHCII⁺CD11c⁻ cells and gating of the CD11b⁻ B cells that are more abundant among donor cells compared to recipient cells (90.6% vs. 1.66%). The far right graph, showing a side by forward scatter plot, demonstrates that these donor MHCII⁺CD11c⁻CD11b⁻ B cells are indeed small cells. (<b>D</b>) Representative flow cytometry plots show the population of MHCII⁻CD11c⁻ cells and gating of the CD11b⁻ T cells that are more abundant among donor cells compared to recipient cells (94.5% vs. 1.11%). The far right graph, showing a side by forward scatter plot, again demonstrates that these donor MHCII⁻CD11c⁻CD11b⁻ T cells are indeed small cells.</p

Splenocyte transfer followed by inhaled LPS leads to pGVHD pathology.

<p>Rag1^−/− mice received allogeneic (Allo) or syngeneic (Syn) splenocytes or no splenocyte transfer (Rag1^−/−NT). Additional wild-type C57BL/6 (B6) mice without splenocyte transfer (B6NT) were used as controls. Allo, Syn, Rag1^−/−NT and B6NT mice underwent daily exposures to aerosolized LPS for 5 days starting 1 week after splenocyte transfer. Mice were euthanized 72 hours after the last LPS exposure. (<b>A</b>) Lung pathology assessment shows perivascular and peribronchiolar mononuclear inflammation in the AlloLPS and SynLPS mice <i>(H&E, 100X)</i>. Only minimal inflammation is seen in AlloNoLPS and SynNoLPS lungs. Rag1^−/−NT mouse lung pathology is shown for additional comparison and is similar to that of B6NT mouse lungs. After LPS, all NT mice have rare mononuclear cells visible in the perivascular and peribronchiolar structures. This is similar to pathology seen in B6NT mice. (<b>B</b>) Lung pGVHD pathology was graded in a blinded fashion using a 0–9 semi-quantitative grading schema to express the thickness of the mononuclear infiltrate around airways and around vessels as well as the overall extent of the pathology in the lung. SynLPS and AlloLPS lungs have a grade of about 8, which is significantly higher than the grade of non-LPS exposed controls where the grade is about 2.5 (AlloLPS vs. AlloNoLPS p = 0.003 and SynLPS vs. SynNoLPS p = 0.0005). As measured by this grading, LPS led to low-grade background inflammation in NT mice as shown in the graph. Data represent the average +/− SEM and **represents p<0.005. Data have been replicated in 3 independent experiments.</p

Allogeneic splenocyte transfer followed by inhaled LPS leads to lung injury and airway hyperreactivity (AHR).

<p>Rag1^−/− mice received allogeneic (Allo) splenocytes or syngeneic (Syn) splenocytes and underwent daily exposures to aerosolized LPS for 5 days starting 1 week after splenocyte transfer. Airway physiology was studied and mice were euthanized 72 hours after the last LPS exposure. (<b>A</b>) BAL fluid protein levels were measured and were found to be significantly higher in AlloLPS mice as compared to SynLPS (p<0.0001). (<b>B</b>) Airway resistance was measured <i>in vivo</i> in response to increasing doses of aerosolized methacholine. Airway resistance in AlloLPS mice was significantly enhanced in dose-related manner as compared to AlloNoLPS and SynLPS controls (p = 0.045). Data represent the average +/− SEM and * = p<0.05, *** = p<0.0005. Data have been replicated in 2 independent experiments.</p