Inhalant exposures to organic dust extract (ODE), lipopolysaccharide (LPS), and peptidoglycan (PGN) induce lung infiltration of CCR2 + monocyte-macrophage (Mɸ) and monocyte subpopulations.
In the first set of experiments, heterozygote CCR2RFP/+ mice were treated once with ODE (25%), LPS (10 µg), PGN (100 µg), or sterile saline with lung tissue cell infiltrates analyzed at 48 h, as a previous study indicated that this was an optimal time point to detect recruited, infiltrating CD11cintCD11bhi transitioning monocytes-macrophages (Mɸ) following acute LPS treatment (12). There were significant increases (p < 0.05) in total cells, CCR2+ cells, and CCR2− cells following ODE, LPS, and PGN as compared to saline control with no difference across the treatment groups (Fig. 1A). The 4 monocyte-macrophage subpopulations including alveolar (Alv) Mɸ (CD11c+CD11blo), activated (Act) Mɸ (CD11c+CD11bhi), transitioning monocyte-Mɸ (CD11cintCD11bhi), and monocytes (CD11c−CD11bhi) were delineated as previously described (12), with representative contour plots shown in Fig. 1B. CCR2 RFP+ and CCR2 RFP− expression in each of the four monocyte/Mɸ subpopulations by treatment condition are depicted in Fig. 1C. CCR2 expression was absent on the Alv Mɸ and Act Mɸ subpopulations but was present on transitioning monocyte-Mɸ and monocyte subpopulations with numbers of these cell subpopulations enumerated in Fig. 1D. The numbers of CCR2+ and CCR2− transitioning monocyte-Mɸ were significantly increased with ODE, LPS, and PGN treatment as compared to saline control (p < 0.05), but the magnitude of the increase was strikingly greater for the CCR2+ (vs. CCR2−) transitioning monocyte-Mɸ cells. There were also significant (p < 0.05) increases in the numbers of CCR2+ and CCR2− monocytes with ODE, LPS, and PGN treatment as compared to saline control with similar magnitude of increases for both CCR2+ and CCR2− monocytes. CCR2− Act Mɸ were increased with ODE, LPS, and PGN vs. saline, and correspondingly, CCR2− Alv Mɸ were decreased with ODE, LPS, and PGN vs. saline (Fig. 1D). Although differences vs. saline were demonstrated, there was no difference in the numbers of the monocytes/Mɸ among ODE, LPS, and PGN. Thus, all environmental exposures examined increased CCR2+ transitioning monocyte-Mɸ and monocyte subpopulations, but there were also increases in CCR2− monocyte subpopulations and to a lesser degree CCR2− transitioning monocyte-Mɸ.
Cell surface expression of Ly6C and F4/80 with monocyte/macrophage (Mɸ) subpopulations following inhalant exposures to ODE, LPS, and PGN.
Cell surface expression of Ly6C, a predominant marker of monocytes and/or associated with pro-inflammatory and pro-fibrotic properties (36) by percent expression and mean fluorescence intensity (MFI) were also investigated and summarized (Fig. 2). Ly6C expression was low (< 5%) on Sal-Alv Mɸ and ODE-, LPS-, PGN-Act Mɸ (data not shown). In contrast, Ly6C percent and MFI expression were increased on both CCR2+ and CCR2− ODE-, LPS-, PGN- induced CD11cintCD11bhi monocyte-Mɸ cells vs. saline control (except MFI expression was not increased for these CCR2− cells following PGN exposure) (Fig. 2A, B). Moreover, Ly6C MFI expression was significantly (p < 0.05) increased on these CCR2+ monocyte-Mɸ cells associated with ODE, LPS, and PGN exposure as compared to the corresponding CCR2− monocyte-Mɸ cells. Ly6C percent expression was high on all monocyte populations with a significant (p < 0.05) increase with LPS-associated CCR2+ and CCR2−monocytes vs. saline (Fig. 2A, B). There was an increase in Ly6C MFI expression with ODE and LPS CCR2− monocytes vs. saline with no difference in intensity of the MFI expression across CCR2 RFP+ monocytes. As observed with transitioning monocyte-Mɸ cells, MFI expression was increased in all CCR2+ monocytes as compared to CCR2− monocytes. These studies demonstrate that Ly6C expression was increased in the recruited CCR2+ cells as well as CCR2− cells following exposure to environmental agents, and as such, Ly6C alone may not discriminate monocyte-macrophage subpopulations.
The monocyte and macrophage marker F4/80 (ADGRE1)(37) was also investigated across cell subpopulations (Supplemental Fig. 2). The percent F4/80 expression was ubiquitous across Sal-Alv Mɸ and ODE-, LPS-, PGN-Act Mɸ subpopulations, but the expression intensity by MFI was increased in the ODE-, LPS-, PGN-Act Mɸ vs. Saline-Alv Mɸ (Supplemental Fig. 2). The percent F4/80 expression was high (~ 60–80%) in the transitioning CCR2+ and CCR2− CD11cintCD11bhi monocyte-macrophage cells (~ 60–80%) and more variable with CD11c−CD11bhi monocytes.
Inhalant exposures to ODE and LPS induce lung infiltration of CCR2 + NK cells and T cells in addition to CCR2− neutrophils, B cells, and T cells.
The number of CCR2+ and CCR2− neutrophils and lymphocytes was also investigated to capture any non-monocyte/macrophage cell-specific CCR2 expression events at 48 h post environmental agent exposure (Fig. 3). CCR2 is recognized to be expressed with NK cells (38) and activated T cells (39). Indeed, there were significant (p < 0.05) increases in CCR2+ NK cells, CD3+CD4+ T cells, and CD3+CD8+ T cells with ODE and LPS but not PGN treatment vs. saline. There were also significant (p < 0.05) increases in CCR2− CD3+CD4+ T cells and CCR2− CD3+CD8+ T cells. ODE, LPS, and PGN treatment did not increase CCR2+ neutrophils or CD19+ B cells. ODE, LPS, and PGN treatment also increased CCR2− neutrophils and CD4+ T cells, and ODE and LPS (but not PGN) increased CCR2− CD8+ T cells.
Inhalant LPS-induced lung inflammatory responses are not reduced in CCR2 knock-out (KO) mice.
Because there were no major differences in ODE-, LPS-, and PGN-induced CCR2+ monocyte-macrophage lung cell infiltrates, LPS was utilized as the prototype environmental inflammatory agent for the remainder of the studies. It was hypothesized that CCR2 knock-out (KO) mice would be protected against LPS-induced lung inflammatory and pro-fibrotic responses due to reduction in the recruitment of transitioning CCR2+ monocyte-Mɸ infiltrates. Although there were significant (p = 0.0005) reductions (54% reduction) in LPS-induced CD11cintCD11bhi monocyte-Mɸ cells, there were no significant reductions in LPS-induced pro-inflammatory and pro-fibrotic mediators in lung homogenates including TNF-α, IL-6, CXCL1, MMP-3, MMP-8, and TIMP-1 (Table 1) and lung histopathology (data not shown) in CCR2 KO mice vs. WT mice. There were also no differences between CCR2 KO and WT mice for LPS-induced total cells, neutrophils, lymphocytes, and macrophages in BALF as well as LPS-induced lung infiltrates including activated Mɸ, monocytes, T and B lymphocytes, and NK cells. In contrast, LPS-induced lung neutrophils were increased in CCR2 KO (vs. WT) mice, and moreover, there were corresponding, likely compensatory, increases in lung and serum CCL2 and CCL7, chemoattractants that predominately drive monocyte recruitment but also can affect lymphocytes and neutrophils, with the LPS treated CCR2 KO mice.
LPS-induced lung transitioning, infiltrating CD11c int CD11b hi are reduced with systemic delivery of clodronate liposomes.
In an alternative approach to deplete recruited lung monocytes-macrophages induced by environmental exposures, intravenous clodronate liposomes (vs. vehicle control liposomes) were dosed one day prior to LPS (and saline) treatment to reduce circulating/systemic reservoir of available monocytes-macrophages with mice euthanized at 48 h following LPS exposure. In these studies, there was a reduction in total lung cells in tissue homogenates associated with a 59% reduction in LPS-induced lung CD11cintCD11bhi monocyte-Mɸ infiltrates in clodronate liposome pre-treated mice compared to control-treated with mice (Fig. 4). In contrast, there were no treatment differences in the number of LPS-induced Alv Mɸ, Act Mɸ or monocytes. There were also no treatment differences in the number of inflammatory cells in BALF and no treatment differences in the number of, CD19+ B cells, CD4+ and CD8+ T cells, and NK cells in tissue homogenates following LPS exposure (Supplemental Table 1).
Effects of systemic delivery of clodronate liposomes with LPS-induced lung inflammation, collagen deposition, and infiltrating CCR2 + cells.
Lung sections from these same mice pre-treated with vehicle and clodronate liposomes followed by saline and LPS challenge were evaluated for histopathological changes by H&E, collagen deposition by trichrome staining, and CCR2+ cell infiltrates (Fig. 5A). Although semi-quantitative inflammatory scores following LPS exposure were reduced with clodronate liposome pre-treatment compared to vehicle control (Fig. 5B), this difference did not reach statistical significance. LPS-induced collagen deposition was reduced by 23% (p < 0.05) with clodronate liposome pretreatment (Fig. 5C). Moreover, LPS-induced CCR2+ cell infiltrates were reduced by 60% (p < 0.05) with clodronate liposome pretreatment (Fig. 5D), consistent with reductions observed in CD11cintCD11b+ monocyte- Mɸ demonstrated by flow cytometry.
LPS-induced lung pro-fibrotic and inflammatory mediators modulated following systemic delivery of clodronate liposomes.
Pre-treatment with intravenous clodronate liposomes (vs. vehicle) also resulted in significant reductions in LPS-induced levels of pro-fibrotic mediators in lung homogenates including MMP-3 (33% reduction), MMP-8 (50% reduction), TIMP-1 (64% reduction), and TGF-β (38% reduction) (Fig. 6). Moreover, there were also significant (p < 0.05) reductions in LPS-induced pro-inflammatory mediators including IL-6 (72% reduction) and neutrophil chemoattractant CXCL1 (57% reduction) with clodronate (vs. vehicle) liposome pre-treatment (Fig. 6). Lung levels of TNF-α induced by LPS exposure were not reduced with clodronate liposome pre-treatment, and there were also no differences in LPS-induced lung CCL2 and CCL7 levels between clodronate and vehicle liposome pre-treatment (Supplemental Table 1).
LPS-induced lung CIT and MAA autoantigens and vimentin expression were reduced with systemic delivery of clodronate liposomes.
Based upon findings of decreased pro-fibrotic mediators and prior findings demonstrating that repetitive inhalant environmental exposures induce post-translational changes implicated in inflammatory and fibrotic lung disease (17, 18), lung tissues were stained for CIT- and MAA-modified antigens as well as vimentin. CIT- and MAA-modified proteins and vimentin were significantly increased following a one-time LPS exposure vs. saline control at 48 h post-LPS exposure (Fig. 7A, B). Moreover, there were significant (p < 0.05) reductions in LPS-induced lung CIT-modified protein expression (39% reduction), MAA-modified protein expression (48% reduction), and vimentin (40% reduction) with clodronate (vs. vehicle) liposome administration.