Environmental exposures

Lipopolysaccharide (LPS) from gram-negative Escherichia coli (O55:B5; Sigma, St. Louis, MO) served as the primary exposure in all experiments. The rationale was that LPS is commercially available and elicits a dose-dependent, reproducible pro-inflammatory lung response in humans and rodents. In studies of monocyte trafficking, comparisons were undertaken using peptidoglycan (PGN) from gram-positive Staphylococcus aureus (Sigma) and an aqueous solution of organic dust extract (ODE) prepared from swine confinement feeding facilities as previously described [26]. Briefly, settled surface dust (1 g) was incubated in sterile Hank’s Balanced Salt Solution (10 mL; Mediatech, Manassas, VA) for 1 h and centrifuged for 30 min at 2850 × g twice, with the final supernate filter-sterilized (0.22 um) to remove microorganisms and coarse particles. Stock ODE was batch prepared and stored at − 20 °C; aliquots were diluted for each experiment to a final concentration (vol/vol) of 25% in sterile phosphate buffered saline (PBS, pH 7.4; diluent). Endotoxin concentrations were determined using the limulus amebocyte lysate assay (Lonza, Walkersville, MD). Endotoxin levels averaged 1.308–2.616 μg (~ 10–50 EU) for 25% ODE. Prior mass spectrometry studies of ODE have revealed significant amounts of muramic acid (peptidoglycan marker) and 3-hydroxy fatty acids (endotoxin marker), but not ergosterol (fungi marker) as compared to house dust [26].

Animal exposure model

C57BL/6 and homozygous CCR2RFP/RFP (B6.129(Cg)-Ccr2tm2.1lfc/J) mice between 6 and 8 weeks of age were purchased from The Jackson Laboratory (Bar Harbor, ME). In this latter strain (#017586; RRID:IMSR_JAX:017586), a monomeric red fluorescent protein (RFP) sequence replaces the coding sequence of the Ccr2 gene, abolishing gene function and thus referred to as CCR2 knockout (KO) mice. Mouse tail snips were collected and shipped for DNA extraction and targeted CCR2 genotyping (TransnetYX, Cordova, TN) to confirm CCR2 KO. To generate heterozygous CCR2+/RFP mice in which CCR2 is functional yet marked by RFP expression, CCR2RFP/RFP mice were bred to C57BL/6 wild-type (WT) mice. For experiments using heterozygous CCR2+/RFP animals, male and female mice were utilized. For the CCR2 KO (and clodronate liposome studies), male mice were utilized, as male mice had increased inflammatory responses with less experimental variability following LPS exposure, consistent with prior studies [12, 27]. Mice were randomized, with AJN, AG, and animal facility staff aware of the randomization, whereas all other authors were blinded. To induce airway inflammation, mice were lightly sedated under isoflurane (VetOne, Boise, ID) and received one treatment with 50 μl of sterile saline (control), ODE (25%), LPS (10 μg), or PGN (100 μg) [28]. Animals were euthanized 48 h following exposure by isoflurane followed by exsanguination (right axillary blood collection). No respiratory distress, signs of stress, or significant weight loss (defined as > 20%) were observed throughout the exposure period.

Clodronate-induced systemic monocyte/macrophage depletion

In separate studies, C57BL/6 WT mice were administered encapsulated clodronate liposomes intravenously to deplete systemic monocytes and recruited monocyte-derived macrophages [29,30,31,32]. Clodronate and control liposomes (Liposoma Technology, Amsterdam, Netherlands; 200 μl × 5 mg/ml) were injected into the tail vein one day prior to LPS and saline control exposure.

Lavage fluid cells and lung homogenates

Bronchoalveolar lavage fluid (BALF) was collected using 3 × 1 mL PBS. Total cell numbers from the combined recovered lavage were enumerated using a BioRad TC 20 cell counter with differential cell counts determined from cytospin-prepared slides (cytopro cytocentrifuge, ELITech Group, Logan, UT) stained with Diff-Quick (Siemens, Newark, DE). Lung tissue homogenates were prepared by homogenizing lung samples (1/2 of right lungs) in 500 μl of sterile phosphate buffered saline (PBS) following removal of BALF and blood from the pulmonary vasculature. From cell-free lung tissue homogenates, levels of TNF-α, IL-6, murine neutrophil chemoattractant CXCL1, murine monocyte (and leukocyte) chemoattractants CCL2 and CCL7, and transforming growth factor (TGF)-β were quantitated by ELISA (R&D Systems, Minneapolis, MN) with minimal detectable difference (MDD) of 1.88, 1.6, 2.0, 0.3, 1.5, 31.3 pg/ml, respectively. Additionally, lung tissue homogenates were assessed for regulators of extracellular matrix deposition including matrix metalloproteinase (MMP)-3 and tissue inhibitor of metalloproteinase (TIMP)-1 (ELISA; R&D Systems; MDD of 0.125 and 0.031 ng/ml, respectively) as well as MMP-8 (ELISA; Abcam, Boston, MA; MDD of 0.053 ng/ml).

Lung cell staining and flow cytometry

Following removal of BALF and blood from pulmonary vasculature, harvested lungs (1/2 of right lungs) were subjected to an automated dissociation procedure using a gentleMACS Dissociator instrument (Miltenyi Biotech, Auburn, CA). Viability of total lung cells was assessed by trypan blue exclusion and a LIVE/DEAD Fixable Blue Dead Cell Stain Kit (Invitrogen, Carlsbad, CA). Cell viability was > 99% with no differences by treatment group(s) (data not shown). Lung cells were incubated with CD16/32 (Fc Block, BioLegend, San Diego, CA) to minimize non-specific antibody staining, then stained with monoclonal antibodies against rat anti-mouse; CD45 (clone: 30-F11; BD Biosciences, Franklin Lake, NJ), CD11b (clone: M1/70; BD Biosciences and BioLegend), Ly6G (clone: 1A8; BD Biosciences), CD11c (clone: N418; Invitrogen), CD4 (clone: RM4-5; BD Biosciences), CD8 (clone: 53–6.7; BD Biosciences), CD19 (clone: 1D3; Invitrogen), hamster anti-mouse CD3e (clone: 145-2C11; BD Biosciences and BioLegend), mouse anti-mouse NK1.1 (clone: PK136; BD Biosciences or BioLegend), Ly6C (clone: HK1.4; BioLegend), and F4/80 (clone: QA17A29; BioLegend or clone: T45-2342; BD Biosciences). Cells were acquired on a BD LSRII Yellow/Green cytometer configured with 355-nm, 405-nm, 488-nm, 561-nm, and 633-nm lasers. Post-acquisition, data were exported and stored using the flow cytometry standard (FCS) 3.1 format and analyzed using FlowJo software version 10.8 (FlowJo, RRID:SCR_008520, Ashland, OR).

The gating strategies for Ly6G+ neutrophils, CD11c+CD11blo alveolar (Alv) macrophages (Mɸ), CD11c+CD11bhi activated (Act) alveolar Mɸ, CD11cintCD11bhi recruited/transitioning monocytes-Mɸ, CD11c−CD11bhi monocytes, CD3+CD4+ T cells, CD3+CD8+ T cells, CD19+ B cells, and NK cells were performed as previously reported [12, 17, 18] with associated RFP+ gating per cell population (Supplemental Fig. 1 and Fig. 1). The percentage of all respective cell populations was determined from live CD45+ lung leukocytes after excluding debris and doublets. This percentage was multiplied by the respective total lung cell numbers to determine specific cell population numbers for each animal.

Fig. 1

Inhalation of organic dust extract (ODE), lipopolysaccharide (LPS), and peptidoglycan (PGN) induce lung CCR2+ monocyte-macrophages (Mɸ) and monocytes. CCR2RFP/+ mice were exposed once to ODE (25%), LPS (10 μg), PGN (100 μg), or saline control and euthanized at 48 h. Scatter plots with bars depict mean with SD delineating cells as total (gray), CCR2+ (green), and CCR2− (red). A Total lung cells enumerated. B Representative contour plot of the four monocyte (mono)-Mɸ subpopulations across groups based upon CD11c and CD11b expression after removal of neutrophils gated from live CD45+ cells after excluding debris and doublets. C RFP ± staining by exposure group and subpopulation. D CD11c+CD11blo alveolar (Alv) Mɸ, CD11c+CD11bhi activated (Act) Mɸ, CD11cintCD11bhi mono-Mɸ, and CD11c−CD11b.hi monocytes determined by multiplying lung cell % population by total lung cells enumerated from lung sample. Statistical analyses were performed with Kruskal–Wallis with Dunn’s test for multiple comparisons (#p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001) vs. respective saline. N = 19 (saline), 9 (ODE), 8 (LPS), 5 (PGN)

Lung histopathology and post-translational modifications

Following removal of BALF and blood from the pulmonary vasculature, left lungs were excised and inflated to 15 cm H2O pressure with 10% formalin (Fisher Scientific, Fair Lawn, NJ) for 24 h to preserve pulmonary architecture [18]. Fixed left lung lobes were then placed into cassettes, embedded in paraffin, cut (4–5 μm) at midpoint sections to include regions of both large and small airways as well as blood vessels, and stained with hematoxylin and eosin (H&E) or preserved for subsequent IHC. H&E-stained slides of entire lung sections from each animal were reviewed at all scanning magnifications and semi-quantitatively scored for the degree and distribution of lung inflammation. Scores were generated by an expert pathologist blinded to treatment conditions utilizing a previously published scoring system (scored 0 to 4) [18, 33] that evaluates the spectrum of inflammatory changes for alveolar and bronchiolar compartments with higher scores indicating greater inflammation.

Lung sections were also stained with modified Masson’s Trichrome and scanned by Aperio scanner (Leica Biosystems, Deer Park, IL). The VENTANA image viewer (version 3.1.4; Roche Diagnostics, Indianapolis, IN) was utilized to capture 10 images per lung section at 20 × from scanned images. Collagen content in trichrome images was quantified as previously described [12, 34] using Image J FIJI plugin (version: 2.9.0/1.53t U.S. National Institutes of Health, Bethesda, MD).

To quantify CCR2+ expression of inflammatory monocyte/macrophages, lung sections were stained with anti-CCR2 (1:100, NBP267700, Lot HM0537, Novus, Littleton, CO) and cross absorbed with donkey anti-rabbit (AlexaFluor488, A21206, Lot #2,156,521, Thermo Fisher, Waltham, MA) at 1:100 and processed as previously described [12]. Slides were mounted with VECTASHIELD® Antifade Mounting Medium with DAPI (4′6-diamindino-2-phenylindole; to identify nuclei)(Cat#H-1200, Lot#ZG1014, Curlingame, CA). Using a Zeiss fluorescent microscope (Zeiss Observer.Z1 Zeiss, White Plains, NY), photographs (10 lung images per mouse) of lung parenchyma were taken, and CCR2+ expression by integrated density was quantified by Image J FIJI plugin.

Citrullinated (CIT) and malondialdehyde acetaldehyde (MAA) modified proteins and vimentin were stained [17]. Increased in the context of inflammatory lung diseases, vimentin is an extracellular matrix protein that is also targeted by post-translational modifications generated during the process of inflammation and increased oxidative stress. Prior studies by our group have demonstrated robust co-localization of MAA and CIT with vimentin in lung tissues of mice and humans with inflammatory arthritis and lung disease [17, 35]. Lung sections were stained with Cy5 rabbit anti-vimentin (Bioss, Woburn, MA, USA, 1:100), Zenon AF 594 label (Invitrogen, Carlsbad, CA, USA) and rabbit polyclonal IgG antibody to MAA [17], or a mouse monoclonal anti-peptidyl-citrulline antibody (clone F95 IgMκ, Millipore Sigma, Burlington, MA, USA). Detection of the F95 antibody was done using an AF 488 AffiniPure donkey anti-mouse IgM, µ chain specific antibody (Jackson Immunoresearch, West Grove, PA, USA). DAPI (4′,6-diamidino-2-phenylindole; to identify nuclei) was added and samples were sealed with Fluormount-G (Southern Biotech, Birmingham, AL, USA). Fluorochromes detected using a Revolve fluorescent microscope (ECHO, San Diego, CA, USA). Images were quantified using ImageJ, and colocalization was performed using the Image J (RRID:SCR_003070) FIJI plugin Coloc 2 [17, 18].

Statistical analysis

Sample-size requirements were extrapolated from previous work assessing post-LPS lung recovery treatments in C57BL/6 [12]. The mean (± SD) of CD11cintCD11bhi transitioning/recruited monocyte-macrophages was 0.26 × 105 (0.09 × 105) with saline and 6.5 × 105 (2.2 × 105) with LPS treatment 48 h post-exposure; thus, a sample size of N = 2 in each group would achieve 80% power at the 0.05 level of significance to determine an influx of these cells following inflammatory agent exposure as compared to saline control. Experimental groups for the CCR2 trafficking studies include at least 2 mice for each group. The maximum sample sizes for the CCR2RFP/+ trafficking studies are N = 19 (saline/Sal), N = 9 (ODE), N = 8 (LPS), and N = 5 (PGN). A sample size of n = 5 would achieve 80% power at the 0.05 level of significance to detect a 60% reduction in CD11cintCD11bhi transitioning/recruited monocyte-macrophages with depletion strategies (i.e., CCR2 KO and clodronate liposomes). For the CCR2 WT vs. KO studies, N = 5 (CCR2 WT-Sal), N = 5 (CCR2 KO-Sal), N = 9 (CCR2 WT-LPS) and N = 9 (CCR2 KO-LPS); and for the clodronate (Clod) versus vehicle (Veh) liposome targeted studies, N = 8 (Veh + Sal), 8 (Clod + Sal), 8 (Veh + LPS), and 9 (Clod + LPS). Experimental groups for those studies include at least 5 mice for each group. Numbers less than the maximum number reflect limitations in available sample quantity or quality.

Data are presented as the mean ± standard deviation (± SD) with scatter plots depicted for each data point. The Shapiro–Wilk test was utilized to test for normality among treatment groups. If the normality condition was satisfied, parametric statistical tests (one-way ANOVA with subsequent Tukey’s multiple comparison test), and if not satisfied, nonparametric statistical (Kruskal–Wallis with subsequent Dunn’s multiple comparison test) were used to assess differences between any two groups. All statistical analyses were performed using GraphPad Prism (version: 10.1.1) software and statistical significance accepted at a p value < 0.05.

Ethics statement

This study was conducted and reported in accordance with ARRIVE guidelines (https://arriveguidelines.org). All animal procedures were also approved by the University of Nebraska Medical Center (UNMC) Institutional Animal Care and Use Committee and were in accordance with the NIH guidelines for the use of rodents.