Lo Re S, Dumoutier L, Couillin I, van Vyve C, Yakoub Y, Uwambayinema F, Marien B, van der Brûle S, van Snick J, Uyttenhove C, Ryffel B, Renauld JC, Lison D, Huaux F.IL-17A-Producing γδ T and Th17 Lymphocytes Mediate Lung Inflammation but Not Fibrosis in Experimental Silicosis. The Journal of Immunology. 2010 Apr 26;184(11):6367–77.


IL-17-producing T lymphocytes play a crucial role in inflammation, but their possible implication in fibrosis remains to be explored. In this study, we examined the involvement of these cells in a  mouse model of lung inflammation and fibrosis induced by silica particles. Upregulation of IL-17A was associated with the development of experimental silicosis, but this response was markedly reduced in athymic, γd T cell-deficient or CD4+ T cell-depleted mice. In addition, γd T lymphocytes and CD4+ T cells, but not macrophages, neutrophils, NK cells or CD8 T cells, purified from the lungs of silicotic mice markedly expressed IL-17A. Depletion of alveolar macrophages or neutralization of IL-23 reduced upregulation of IL-17A in the lung of silicotic mice. IL-17R–deficient animals (IL-17R-/- ) or IL-17A Ab neutralization, but not IL-22 -/- mice, developed reduced neutrophil influx and injury during the early lung response to silica. However, chronic inflammation, fibrosis, and TGF-β expression induced by silica were not attenuated in the absence of IL-17R or -22 or after IL-17A Ab blockade. In conclusion, a rapid lung recruitment of IL-17A–producing T cells, mediated by macrophage-derived IL-23, is associated with experimental silicosis in mice. Although the acute alveolitis induced by silica is IL-17A dependent, this cytokine appears dispensable for the development of the late inflammatory and fibrotic lung responses to silica. [/toggle_content] [custom_table]

Clodronate Concentration Total Lipid Concentration Lipid Composition Lipid Mole % Liposome Type Control Liposomes
5 mg/ml 23.5 mg/ml EPC/Chol 86/14 MLV PBS
[/custom_table] [custom_table]
Animal Description Clodronate Dose Dosing Method/Site Target Phagocytes Systemic Dosing? Systemic Results
C57BL/6 mice2 100 µl pharyngeal aspiration / lungs alveolar macrophages (AM) no not evaluated

2Base or background strain. Variants, mutants or otherwise genetically altered strains were also used in this study.



  1. No details on pharyngeal dosing technique published.
  2. Citations related to macrophage depletion compare intratracheal and aerosol administration methods, not pharyngeal administration.


  1. >80% of the AM were depleted (BAL cell counts via light microscopy) 3 days post-treatment (data not shown).
  2. Complete inhibition of IL-7A production is only other depletion-related data published.




Ivanov S, Fontaine J, Paget C, MachoFernandez E, Maele LV, Renneson J, Malliet I, Wolf NM, Rial A, Léger H, Ryffel B, Frisch B, Chabalgoity J, Sirard JC, Benecke A, Faveeuw C, Trottein F.
Key role for respiratory CD103+ dendritic cells, IFN-γ and IL-17 in protection against Streptococcus pneumoniae infection in response to α-galactosylceramide.
J Infect Dis. [Internet]. 2012 Jun 21 [cited 2012 Jun 27]; Available from: http://jid.oxfordjournals.org/content/early/2012/06/21/infdis.jis413
[toggle_content title=”Abstract”] Background. Exogenous activation of pulmonary invariant Natural Killer T (iNKT) cells, a population of lipid-reactive αβ T lymphocytes, by means of mucosal α-galactosylceramide (α-GalCer) administration, is a promising approach to control respiratory bacterial infections. We undertook the present study to characterize mechanisms leading to α-GalCer-mediated protection against lethal infection with S. pneumoniae serotype 1, a major respiratory pathogen in humans.
Methods and Results. α-GalCer was administered by the intranasal route before infection with S. pneumoniae. We showed that respiratory dendritic cells (DCs), most likely the CD103+ subset, play a major role in the activation (IFN-γ and IL-17 release) of pulmonary iNKT cells, whilst alveolar and interstitial macrophages are minor players. After challenge, S. pneumoniae was rapidly (4h) eliminated in the alveolar spaces, a phenomenon that depended on respiratory DCs and neutrophils, but not macrophages, and on the early production of both IFN-γ and IL-17. Protection was also associated with the synthesis of various interferon-dependent and IL-17-associated genes as revealed by transcriptomic analysis.
Conclusions. These data imply a new function for pulmonary CD103+ DCs in mucosal activation of iNKT cells and establish a critical role for both IFN-γ and IL-17 signalling pathways in mediating the innate immune response to S. pneumoniae.[/toggle_content] [toggle_content title=”Clodronate Liposome Parameters”] [custom_table]
Clodronate Concentration Total Lipid Concentration Lipid Composition Lipid Mole % Liposome Type Control Liposomes
10 mg/ml 1 46 mg/ml EPC/Chol 86/14 MLV none

1 Clodronate and lipid concentrations assumed based on information in paper (dosed 2 mg in 0.2 ml).

[/custom_table] [/toggle_content] [toggle_content title=”Animals and Dosing”] [custom_table]
Animal Description Clodronate Dose Dosing Method/Site Target Phagocytes Adjunct Dosing? Adjunct Dosing Route
C57BL/6, CD11c mice2, male, 8 w 20 µg intranasal instillation alveolar no NA

2Base strains; mutants or transgenic animals also used.

[/custom_table] [/toggle_content] [toggle_content title=”Notes”]
  1. Clodronate and lipid concentrations assumed based on referenced paper, although references provide little information as to final clodronate concentrations.
  2. Reference for clodronate liposome preparation — van Rooijen N, van Nieuwmegen R. Elimination of phagocytic cells in the spleen after intravenous injection of liposome-encapsulated dichloromethylene diphosphonate. An enzyme-histochemical study. Cell Tissue Res. 1984;238(2):355–8.
[/toggle_content][toggle_content title=”Results”]
  1. ~74% of the alveolar macrophages collected by lavage were depleted 48 h post-administration of clodronate liposomes; counts at other time points were not taken.
  2. Alveolar macrophage depletion did not affect pulmonary CFU at 4 h post-infection or mortality rate.


Kooguchi K, Hashimoto S, Kobayashi A, Kitamura Y, Kudoh I, Wiener-Kronish J, Sawa,T.
Role of alveolar macrophages in initiation and regulation of inflammation in Pseudomonas aeruginosa pneumonia.
Infect. Immun. 1998 Jul;66(7):3164–9.
[toggle_content title=”Abstract”] To evaluate the role of alveolar macrophages (AMs) in acute Pseudomonas aeruginosa pneumonia in mice, AMs were depleted by aerosol inhalation of liposomes containing clodronate disodium. AM-depleted mice were then intratracheally infected with 5 x 105 CFU of P. aeruginosa . In addition to monitoring neutrophil recruitment and chemokine releases, lung injury was evaluated soon after infection (8 h) and at a later time (48 h). At 8 h, depletion of AMs reduced neutrophil recruitment, chemokine release, and lung injury. At 48 h, however, depletion of AMs decreased bacterial clearance and resulted in delayed movement of neutrophils from the site of inflammation with aggravated lung injury. With instillation of 5 x 107 CFU of bacteria, AM-depleted mice showed low mortality within 24 h of infection but high mortality at a later time, in contrast to non-AM-depleted mice. These results demonstrate that depletion of AMs has beneficial early effects but deleterious late effects on lung injury and survival in cases of P. aeruginosa pneumonia.
[/toggle_content] [toggle_content title=”Clodronate Liposome Parameters”] [custom_table]
Clodronate Concentration Total Lipid Concentration Lipid Composition Lipid Mole % Liposome Type Control Liposomes
not stated 11 mg/ml EPC/Chol 57/43 REV none
[/custom_table] [/toggle_content] [toggle_content title=”Animals and Dosing”] [custom_table]
Animal Description Clodronate Dose Dosing Site/Method Target Phagocytes Systemic Dosing? Systemic Results
CD-1, male, 35-37 g not stated aerosol / lungs alveolar macrophages (AM) no not evaluated
[/custom_table] [/toggle_content] [toggle_content title=”Notes”]
  1. Authors state that liposomes were prepared as in Publication 1, however lipid composition is different as is final volume. Encapsulated clodronate is not published. Lipid concentration calculated assuming 100% recovery.
  2. As discussed for publication 1., passing liposomes through a syringe filter is not equivalent to extrusion.
  3. Aerotech II nebulizer at 12 L/min. No further details published.
[/toggle_content] [toggle_content title=”Results”]
  1. >95% of the AM were depleted (BAL cell counts via light microscopy).
  2. No neutrophils at t=0 post-innoculation, therefore neutrophilia is not observed in this model.
  3. No control liposomes dosed; could chemokine levels be altered by liposomes alone? Not likely but important control.


Hashimoto S, Pittet JF, Hong K, Folkesson H, Bagby G, Kobzik L, Frevert C, Wanatabe K, Tsurufuji S, Weiner-Kronish, J.
Depletion of alveolar macrophages decreases neutrophil chemotaxis to Pseudomonas airspace infections.
American Journal of Physiology – Lung Cellular and Molecular Physiology. 1996 May 1;270(5):L819 –L828.
[toggle_content title=”Abstract”] The mechanism for neutrophil (PMN) influx into infected airspaces of the lung is not known. To determine whether alveolar macrophage products are important in the initiation of chemotaxis, we depleted rats of alveolar macrophages by aerosolizing negatively charged oligolamellar liposomes complexed to clodronate disodium. Ninety-five percent of the alveolar macrophages were depleted, and lung injury and inflammation were minimized with this depletion technique. Rats depleted of alveolar macrophages were then anesthetized, and either 5 x 106 colony-forming units (CFU) or 5 x 107 CFU of Pseudomonas aeruginosa were instilled into the airspaces of these animals. When recombinant macrophage inflammatory protein (MIP-2) was intratracheally instilled into rats depleted of alveolar macrophages, PMN were recruited to their airspaces. Nonetheless, PMN numbers were decreased in the lavage fluids when moderate or large inoculums of bacteria were instilled into depleted rats, although the PMN response appeared dose dependent. Levels of bioactive tumor necrosis factor-α and immunoreactive proteins CINC/gro (cytokine-induced PMN chemoattractant) in the lavage fluids obtained from infected rats depleted of alveolar macrophages were significantly decreased compared with the levels in the lavage fluids obtained from normal infected rats. MIP-2 mRNA expression, as detected by Northern analysis, was also decreased in the infected lungs of depleted rats, and the lavage fluid from these rats had significantly decreased chemotactic activity. Therefore these results suggest that alveolar macrophage products play a direct role in the initial recruitment of PMN into infected lungs. [/toggle_content] [toggle_content title=”Clodronate Liposome Parameters”] [custom_table]
Clodronate Concentration Total Lipid Concentration Lipid Composition Lipid Mole % Liposome Type Control Liposomes
7 mg/ml 33.4 mg/ml EPC/BPS/Chol 55/9/36 REV PBS(-)
[/custom_table] [/toggle_content] [toggle_content title=”Animals and Dosing”] [custom_table]
Animal Description Clodronate Dose Dosing Site/Method Target Phagocytes Systemic Dosing? Systemic Results
Sprague Dawley rats, male, 300-375 g 0.05-0.25 µmoles aerosol / lungs alveolar macrophages (AM) no no change in peripheral moncytes or PMN detected
[/custom_table] [/toggle_content] [toggle_content title=”Notes”]
  1. The authors state that liposomes were “extruded” through 0.2 µm syringe filters. A syringe filter is not a substitute for extrusion therefore the resulting size distribution is questionable. Liposomes >400 nm have been shown to be disrupted by aerosolization, therefore it’s likely that there was free clodronate in the aerosol.
  2. Aerotech II nebulizer at 10 L/min. MMAD = 1.6 µm; GSD = 2.5. Fluorescent liposome aerosol delivery experiments indicated that 0.1-0.5% of the dose was delivered to the lung. This value was used to estimate the clodronate dose delivered to the lungs. The authors estimate that 26X more drug is delivered by this method than by liquid installation.
[/toggle_content] [toggle_content title=”Results”]
  1. >95% of the AM were depleted (BAL cell counts via light microscopy).
  2. The authors further state that clodronate liposome delivery by aerosol prevents neutrophilia often observed when direct instillation is used.