LPS-induced lung inflammation in marmoset monkeys - an acute model for anti-inflammatory drug testing

Abstract

Increasing incidence and substantial morbidity and mortality of respiratory diseases requires the development of new human-specific anti-inflammatory and disease-modifying therapeutics. Therefore, new predictive animal models that closely reflect human lung pathology are needed. In the current study, a tiered acute lipopolysaccharide (LPS)-induced inflammation model was established in marmoset monkeys (Callithrix jacchus) to reflect crucial features of inflammatory lung diseases. Firstly, in an ex vivo approach marmoset and, for the purposes of comparison, human precision-cut lung slices (PCLS) were stimulated with LPS in the presence or absence of the phosphodiesterase-4 (PDE4) inhibitor roflumilast. Pro-inflammatory cytokines including tumor necrosis factor-alpha (TNF-α) and macrophage inflammatory protein-1 beta (MIP-1β) were measured. The corticosteroid dexamethasone was used as treatment control. Secondly, in an in vivo approach marmosets were pre-treated with roflumilast or dexamethasone and unilaterally challenged with LPS. Ipsilateral bronchoalveolar lavage (BAL) was conducted 18 hours after LPS challenge. BAL fluid was processed and analyzed for neutrophils, TNF-α, and MIP-1β. TNF-α release in marmoset PCLS correlated significantly with human PCLS. Roflumilast treatment significantly reduced TNF-α secretion ex vivo in both species, with comparable half maximal inhibitory concentration (IC(50)). LPS instillation into marmoset lungs caused a profound inflammation as shown by neutrophilic influx and increased TNF-α and MIP-1β levels in BAL fluid. This inflammatory response was significantly suppressed by roflumilast and dexamethasone. The close similarity of marmoset and human lungs regarding LPS-induced inflammation and the significant anti-inflammatory effect of approved pharmaceuticals assess the suitability of marmoset monkeys to serve as a promising model for studying anti-inflammatory drugs.

Figure 1. Study design of LPS-induced lung inflammation in marmoset monkeys.

Animals were stimulated intrabronchially with 500 ng LPS instilled unilaterally after 5-day oral pre-treatment with either roflumilast (7 µg/kg bw) or dexamethasone (2 mg/kg bw). Sham-treated individuals receiving water were used as positive controls. Unilateral bronchoalveolar lavage (BAL) was performed 18 hours after LPS provocation. BAL fluid was analyzed for neutrophils, TNF-α and MIP-1β secretion. Baseline levels were determined three weeks before the first treatment and served as control.

Figure 2. LPS-dependent increase in cytokines and chemokines ex vivo.

Ascending TNF-α (A) and intracellular MIP-1β (B) production after 24-hour incubation with increasing concentrations of LPS in marmoset PCLS. LPS-induced increase in TNF-α (C) and intracellular MIP-1β (D) in marmoset PCLS was significantly suppressed by dexamethasone (dxm). Marmoset PCLS and marmoset WBA (E) on the one hand and marmoset PCLS and human PCLS (F) on the other hand showed significant correlations for TNF-α secretion (Spearman’s rank correlation coefficient (r_S)  = 1.0 with p  = 0.0004, and r_s  = 0.9 with p  = 0.01, respectively). Symbols: ○: 2.5 ng/mL LPS, □: 5 ng/mL LPS, ▾: 10 ng/mL LPS, ▵: 100 ng/mL LPS, ◊: 250 ng/mL LPS, x: 500 ng/mL LPS. Data are presented as mean ± SEM, *p<0.05, **p<0.01, Mann-Whitney test (TNF-α: n  = 6, MIP-1β: n  = 4). Correlations were evaluated using a linear regression analysis model combined with Spearman’s rank correlation coefficient. NHP  =  non-human primate, int  =  intracellular.

Figure 3. Dose-response curves of roflumilast in marmoset and human PCLS.

Marmoset and human PCLS were incubated with 100 ng/mL LPS and increasing concentrations of roflumilast (rof, 0.6–310 nM) for 24 hours. The half maximal inhibitory concentrations were detected at 1.3 nM for marmoset and 1.1 nM for human (dashed lines). Data are presented as mean ± SEM, marmoset: n  = 5; human: n  = 4. TNF-α concentration in supernatants was determined by ELISA. Marmoset  =  grey symbols, human  =  black symbols.

Figure 4. Changes in absolute cell numbers in bronchoalveolar lavage (BAL) fluid after LPS challenge.

Sham-treated, dexamethasone (dxm)-treated, and roflumilast (rof)-treated marmosets were intrabronchially challenged with 500 ng LPS. Eighteen hours later, ipsilateral BAL was performed. Total cells (A), neutrophils (B), macrophages (C), and lymphocytes (D) were differentiated and quantified using light microscopy after Pappenheim staining. Data are presented as scatter dot plot with median, *p<0.05, ***p<0.001, one-tailed Mann-Whitney test against sham.

Figure 5. Changes in relative cell numbers in bronchoalveolar lavage (BAL) fluid after LPS challenge.

Sham, dexamethason (dxm) and roflumilast (rof)-treated marmosets were intrabronchially challenged with 500 ng LPS. Eighteen hours later, an ipsilateral BAL was performed. Relative numbers of neutrophils (A), macrophages (B), and lymphocytes (C) were determined. Cells were quantified and differentiated using light microscopy after Pappenheim staining. Data are presented as scatter dot plot with median; *p<0.05, ***p<0.001, one-tailed Mann-Whitney test against sham.

Figure 6. LPS-induced changes in bronchoalveolar lavage (BAL) fluid.

Representative cytospots of BAL at x200 original magnification after Pappenheim staining. (A) Macrophages are the predominant cell type in unchallenged lung lobes. (B) LPS challenge induced strong neutrophilic influx in sham-treated animals. This effect could be significantly attenuated by (C) roflumilast and (D) dexamethasone pre-treatment. Scale bar  = 50 µm.

Figure 7. Analysis of bronchoalveolar lavage (BAL) fluid.

TNF-α content in BAL fluid of marmoset monkeys was significantly reduced in dexamethasone-treated (dxm, p = 0.04) and roflumilast-treated (rof, p = 0.049) animals in contrast to sham-treated individuals (A). MIP-1β concentrations in BAL fluid were reduced by trend in both treatment groups (sham vs. dxm: p = 0.27; sham vs. rof: p = 0.17) (B). Data are presented as scatter dot plot with median, *p<0.05, ***p<0.0.01, one-tailed Mann-Whitney test against sham.