Role of Arginase 1 from Myeloid Cells in Th2-Dominated Lung Inflammation

<div><p>Th2-driven lung inflammation increases Arginase 1 (Arg1) expression in alternatively-activated macrophages (AAMs). AAMs modulate T cell and wound healing responses and Arg1 might contribute to asthma pathogenesis by inhibiting nitric oxide production, regulating fibrosis, modulating arginine metabolism and restricting T cell proliferation. We used mice lacking Arg1 in myeloid cells to investigate the contribution of Arg1 to lung inflammation and pathophysiology. In six model systems encompassing acute and chronic Th2-mediated lung inflammation we observed neither a pathogenic nor protective role for myeloid-expressed Arg1. The number and composition of inflammatory cells in the airways and lungs, mucus secretion, collagen deposition, airway hyper-responsiveness, and T cell cytokine production were not altered if AAMs were deficient in Arg1 or simultaneously in both Arg1 and NOS2. Our results argue that Arg1 is a general feature of alternative activation but only selectively regulates Th2 responses. Therefore, attempts to experimentally or therapeutically inhibit arginase activity in the lung should be examined with caution.</p></div

Arg1 expression by macrophages does not regulate acute schistosome egg-induced pulmonary granuloma formation and Th2 response.

<p>Mice deficient in Arg1, iNOS, or both enzymes were sensitized by i.p. injection of eggs, then challenged by a single i.v. injection of eggs to form pulmonary granulomas and analyzed at 2 weeks. A) T lymphocytes (CD3+), macrophages (F4/80+), and Arginase1+ cells were detected in fixed lung serial sections using immunohistochemistry. Images represent 8 mice of each genotype. B) The volume and eosinophil composition of 10–30 granulomas per mouse were scored in Geimsa-stained lung sections. C) Collagen deposition was compared by measuring L-hydroxyproline in matched lobes and calculating, by mass, total lung content. D) Expression of Arginase 2 or E) IL-13 mRNA and the Th2-responsive genes RELM-α, Mucin 5AC, and Gob5 were compared in lung tissue by quantitative PCR, normalized to the mean naïve WT level. F) To evaluate CD4 T cell responses, lung leukocytes were restimulated with PMA plus ionomycin and stained to detect IL-13, IL-4, IL-5, and IFN-γ by flow cytometry. No significant differences were observed between the challenged groups in B) to F). Results were combined from two independent experiments totaling 11 to 19 mice per group, plus 6 naïve WT controls. Individual mice and group means are shown.</p

Arg1 expression by macrophages does not alter chronic SEA-induced airway inflammation.

<p>Arg1 KO mice were sensitized to schistosome eggs then treated with 8 weekly intra-tracheal challenges of soluble egg antigens to provoke chronic airway inflammation and analyzed 1 day after the final dose. A) The intensity and distribution of mucus (AB-PAS) and collagen (picrosirius red) were compared by histology in stained lung sections. Representative images of mucus-producing airway epithelium and underlying collagen deposition are shown. B) Mucus was visualized by AB-PAS staining and scored on a 0–5 point scale. C) Total lung collagen deposition was measured using L-hydroxyproline content. D) Induction of RELM-α, Mucin 5AC, and Gob5, and mRNA in lung tissue was compared by quantitative PCR, normalized to mean expression in naïve mice. E) Leukocytes from the airways were recovered by broncho-alveolar lavage and categorized by cytospin analysis. Cell numbers did not differ between WT and Arg1 KO mice (data not shown). F) Lung and airway leukocytes were restimulated with PMA plus ionomycin, stained, and analyzed by flow cytometry to compare cytokine production by CD4+ T lymphocytes. No significant differences were observed between challenged WT and Arg1 KO mice. Results were combined from two independent experiments totaling 12 WT and 7 Arg1 KO naïve mice, and 15 WT and 12 Arg1 KO challenged mice. Individual mice and group means are shown.</p

Arg1 expression by macrophages does not affect the outcomes of ovalbumin or Aspergillus-induced allergic airway and lung inflammation.

<p>A) WT and Arg1 KO mice were sensitized and challenged with intranasal Ova, then tested for airway hyper-reactivity. B) Airway-infiltrating leukocytes were recovered by lavage, counted, and categorized by cytospin analysis. C) Ova-specific serum IgE levels were compared by ELISA. D, E, F) Equivalent experiments compared the allergic response to Aspergillus. G) Aspergillus-induced lung inflammation was evaluated for eosinophil recruitment in eosin-stained sections and mucus production in PAS-stained sections. Representative histological images are shown. Except for the numbers of ovalbumin-induced inflammatory airway leukocytes, no significant differences were observed between challenged WT and Arg1 KO mice. Data represent 1 to 3 independent experiments including >8 mice/group, detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061961#pone-0061961-t001" target="_blank">Table 1</a>. Group means and standard deviations are shown.</p

Arg1 expression by macrophages does not regulate chronic schistosome egg-induced pulmonary granuloma formation and Th2 response.

<p>Control and Arg1 KO mice were sensitized by i.p. injection of eggs, then challenged with 5 weekly doses of intravenous eggs to repeatedly form pulmonary granulomas and analyzed 2 weeks after the final challenge. A) The intensity and location of mucus (AB-PAS) and collagen (picrosirius red) were compared by histology in stained lung sections. Representative images of strong mucus production in airways adjacent to granulomas and dense collagen deposition around the perimeters of granulomas are shown. B) The volume and eosinophil composition of 6–29 granulomas per mouse were scored in Geimsa-stained lung sections. C) Cell-associated mucus around the airways was detected by AB-PAS staining and scored on a 0–5 point scale. D) Total lung collagen deposition was assessed by measuring L-hydroxyproline. E) Induction of mRNA transcription for the Th2-responsive genes RELM-α, Mucin 5AC, and Gob5 and was tested in lung tissue by quantitative PCR, normalized to mean expression in naïve WT mice. F) Arginase enzymatic activity was measured in lung tissue samples from naïve control and egg challenged mice. No significant differences were observed between challenged WT and Arg1 KO mice in A) to E). Results are from one experiment including 9 WT and 11 Arg1 KO challenged mice, plus 5 WT and 3 Arg1 KO naïve controls. Individual mice and group means are shown.</p

Macrophage populations in livers of <i>S. mansoni</i>-infected IL-4Rα^flox/ΔLysM^Cre mice express <i>Il4r</i>α and alternative activation markers.

<p>IL-4Rα^flox/ΔLysM^Cre mice (open bars) and IL-4Rα^flox/Δ littermate controls (solid bars) were infected percutaneously with 35 cercariae. From mice infected for 9 weeks, CD45+ SiglecF- CD11b+ Ly6G- F4/80+ CD64+ liver leukocytes were sorted and separated based on Ly6C expression with a flow cytometer. Gene expression was measured by qPCR (n = 3; *p<0.05, ***p<0.001). Fold change is displayed relative to gene expression from CD45+ SiglecF- CD11b+ Ly6G- F4/80+ CD64+ Ly6C+ cells sorted from naïve IL-4Rα^flox/Δ littermate control livers. Data shown are mean ±SEM and represent at least two independent experiments.</p

A population of inflammatory IL-4Rα-expressing myeloid cells resists LysM^Cre-mediated deletion.

<p>BALB/c, IL-4Rα^flox/Δ, and IL-4Rα^flox/ΔLysM^Cre mice were injected i.p. with 2 ml thioglycollate 4 d prior to harvest or were left untreated (naïve). Peritoneal cells were harvested from each group, stimulated for 30 min with 20 ng/ml IL-4 (black outline), and compared to unstimulated cells (solid gray). IL-4Rα function was assessed by IL-4-induced phosphorylation of STAT6 using flow cytometry. A, D. Gating strategy for lymphocytes and F4/80^hi CD11b^hi macrophages. Detection of pSTAT6 in lymphocytes (B, E) and macrophages (C, F). Each histogram peak represents an individual mouse (n = 2–6 for 2 independent experiments). G. DNA was isolated from F4/80^hi CD11b^hi macrophages FACS sorted from naïve and thioglycollate-treated IL-4Rα^flox/flox, IL-4Rα^flox/Δ, IL-4Rα^flox/ΔLysM^Cre and IL-4Rα^Δ/Δ mice. Rearrangement of the <i>Il4rα</i> locus was measured using PCR to compare the presence of wild-type (WT) and knockout (KO) <i>Il4rα</i> alleles. Quantification of band intensity is shown in the right panels. Aggregate intensity of WT product plus KO product was normalized to 100 percent for each sample. H. In a separate experiment, CD11b⁺ F4/80⁺ macrophages were sorted from the same naïve or thioglycollate-elicited peritoneal cells. <i>Lyz2</i> gene expression was found to be lower in thioglycollate-elicited macrophages than naïve macrophages (n = 2–5 for 2 independent experiments). Data shown are mean ±SEM and represent two independent experiments (*p<0.05).</p

Inflammation but not fibrosis is exacerbated in chronically infected IL-4Rα^flox/ΔLysM^Cre mice.

<p>IL-4Rα^flox/ΔLysM^Cre mice and IL-4Rα^flox/Δ littermate controls were infected percutaneously with 35 <i>S. mansoni</i> cercariae. A–C. Representative 10× images of granuloma formation 9 weeks and 16 weeks post-infection from (A) hematoxylin and eosin-stained sections of intestinal tissue, (B) Giemsa-stained sections of liver tissue, or (C) picrosirius red-stained sections of liver tissue. D. Liver granuloma size in IL-4Rα^flox/ΔLysM^Cre (open bars) and IL-4Rα^flox/Δ littermate control (solid bars) mice. E. Liver fibrosis was assessed by hydroxyproline content, normalized to mass or worm pairs recovered by perfusion of infected mice through the portal vein. Data shown are mean ±SEM and represent two independent experiments (n = 15, ns = not significant).</p

Normal cytokine response in <i>S. mansoni</i>-infected IL-4Rα^flox/ΔLysM^Cre mice.

<p>IL-4Rα^flox/ΔLysM^Cre mice and IL-4Rα^flox/Δ littermate controls were infected percutaneously with 35 <i>S. mansoni</i> cercariae. A. Serum IL-13Rα2 levels were measured by ELISA 9 or 16 weeks post-infection. The open and solid portions of each bar correspond to unbound IL-13Rα2 and IL-13Rα2 bound to IL-13, respectively. B. Tissue cytokine levels. Expression of <i>il12p40</i> and <i>il10</i> was quantified by qPCR from liver tissue snips of IL-4Rα^flox/ΔLysM^Cre mice (open bars) or IL-4Rα^flox/Δ littermate controls (solid bars). C. Th2 response. Liver leukocytes were isolated from IL-4Rα^flox/ΔLysM^Cre mice (open bars) or IL-4Rα^flox/Δ littermate controls (solid bars), stimulated with phorbol myristate acetate/ionomycin, and analyzed by flow cytometry. The percentage of CD4⁺ leukocytes expressing intracellular IL-4 and IL-13 are shown. (n = 7-15 for each experiment, ns = not significant). Data shown are mean ±SEM and represent two independent experiments.</p

<i>Lyz2</i>^lo macrophages develop features of AAMs in response to <i>S. mansoni</i> eggs.

<p>IL-4Rα^flox/ΔLysMCre mice (open bars) and littermate controls (solid bars) were left untreated (naïve), challenged with 5000 <i>S. mansoni</i> eggs i.p. 4 days before harvest (1^o), 18 days before harvest (1^o-rested), or challenged on both 18 days and 4 days before harvest (1^o-rechallenged). A. Total peritoneal cells were sorted for F4/80^hi CD11b^hi cells at a purity of >90%. B. Representative 20× images of sorted F4/80^hi CD11b^hi macrophages after cytospin and hematoylin and eosin staining. C,D. The sorted cells were assayed for <i>Il4rα</i> and <i>Lyz2</i> gene expression (C), and gene expression of markers of alternative activation (D). Fold change in gene expression is shown relative to the expression levels in sorted F4/80^hi CD11b^hi cells from naïve littermate controls. E. Surface expression of mannose receptor measured by flow cytometry on unsorted F4/80^hi CD11b^hi peritoneal cells from the same treatment groups. F. Arginase activity in sorted macrophages. (n = 3–6, ns = not significant) Data shown are mean ±SEM and represent at least two independent experiments.</p