Distinct Tlr4-expressing cell compartments control neutrophilic and eosinophilic airway inflammation - PubMed (original) (raw)

Distinct Tlr4-expressing cell compartments control neutrophilic and eosinophilic airway inflammation

J W McAlees et al. Mucosal Immunol. 2015 Jul.

Abstract

Allergic asthma is a chronic, inflammatory lung disease. Some forms of allergic asthma are characterized by T helper type 2 (Th2)-driven eosinophilia, whereas others are distinguished by Th17-driven neutrophilia. Stimulation of Toll-like receptor 4 (TLR4) on hematopoietic and airway epithelial cells (AECs) contributes to the inflammatory response to lipopolysaccharide (LPS) and allergens, but the specific contribution of TLR4 in these cell compartments to airway inflammatory responses remains poorly understood. We used novel, conditionally mutant Tlr4(fl/fl) mice to define the relative contributions of AEC and hematopoietic cell Tlr4 expression to LPS- and allergen-induced airway inflammation. We found that Tlr4 expression by hematopoietic cells is critical for neutrophilic airway inflammation following LPS exposure and for Th17-driven neutrophilic responses to the house dust mite (HDM) lysates and ovalbumin (OVA). Conversely, Tlr4 expression by AECs was found to be important for robust eosinophilic airway inflammation following sensitization and challenge with these same allergens. Thus, Tlr4 expression by hematopoietic and airway epithelial cells controls distinct arms of the immune response to inhaled allergens.

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Figures

Figure 1

Figure 1. Generation of a _Tlr4_fl/fl mouse

(a) Structure of the targeting vector and (b) strategy used to create Tlr4fl/fl mice. (c) PCR-based assay confirming selective deletion of Tlr4 from genomic DNA from the indicated cell types; total splenocytes, Spl; bone marrow, BM; and airway epithelial cells, AECs. Section 1, _Tlr4_fl/fl; Section 2, _Tlr4_fl/flVav1-Cre; Section 3, _Tlr4_fl/fl; Section 4, _Tlr4_fl/flShh-Cre. Tlr4 mRNA levels in (d) AECs and (e) total splenocytes. Data shown represent mean values ±SEM, and are from one experiment, representative of 2–3. N = 4 to 6 mice per group. ***P<0.001.

Figure 2

Figure 2. LPS-responsiveness of cells from _TLR4fl/fl_Shh-Cre and _TLR4fl/fl_Vav1-Cre mice

(a) TNFα production by LPS- and Pam3Cys-treated BMDCs (_n_=4–6 mice). (b) CCL20 production by LPS-, HDM- and Pam3Cys-treated AECs (_n_=6–10 mice/group). (c) Serum TNFα in mice 24 h after i.p. injection of LPS (_n_=6–8 mice/group). Data represent mean values ±SEM from one of 2–3 experiments yielding similar results. *P<0.05; **P<0.01; ***P<0.001.

Figure 3

Figure 3. _Tlr4_-expressing hematopoietic cells promote LPS-mediated neutrophilic inflammation

(a) Time course of neutrophil recruitment following inhalation of 1 μg LPS. (b) LPS dose-response for neutrophils in mice harvested 24 h after treatment. Chemokine and cytokine levels in the BAL fluid at (c) 2 h and (d) 24 h after treatment with 1 μg LPS (n.d., not detectable). Data represent mean values ±SEM from one experiment, representative of three, with 4–12 mice per group. *P<0.05; **P<0.01; ***P<0.001.

Figure 4

Figure 4. Role of _Tlr4_-expressing AECs and hematopoietic cells in innate immune responses to HDM

(a) Neutrophils in BAL fluid of mice 24 h after a single instillation of HDM. (b) CXCL1 and CXCL2 and (c) IL-25 and IL-33 levels in the BAL fluid. Data represent means ±SEM from one experiment, representative of two, with 6–8 mice per group. *P<0.05; **P<0.01.

Figure 5

Figure 5. _Tlr4_-expressing AECs and hematopoietic cells control distinct aspects of HDM-driven allergic airway inflammation

Airway inflammation in the HDM model of asthma. (a) Mean cell numbers ±SEM are shown for the indicated cell types in BAL fluid from WT and _Tlr4_−/− mice. Data are from one experiment, representative of two. (b) _Tlr4_fl/flShh-cre and (c) _Tlr4_fl/flVav1-cre mice sensitized and challenged with HDM extracts. Values for the indicated cell types represent mean cell numbers ±SEM from one experiment, representative of three. N = 6 –12 mice/group. *P<0.05; **P<0.01; ***P<0.001.

Figure 6

Figure 6. Effect of _Tlr4_-expressing cell types on cytokine and chemokine production in the lung

Cytokines and chemokines in the supernatants of whole lungs restimulated ex vivo for 5 days with HDM from (a) _Tlr4_fl/flShh-cre and (b) _Tlr4_fl/flVav1-cre mice. Data represent mean concentrations ±SEM from one experiment, representative of two. N = 6 to 12 mice/group. *P<0.05.

Figure 7

Figure 7. Allergic responses to HDM sensitization and challenge in bone marrow chimeric mice

(a) Airway inflammation and (b, c) cytokine production in the HDM model of asthma. Cytokines are from cultures of whole lungs restimulated ex vivo for 5 days with HDM. Data represent mean values ±SEM from one experiment, representative of three. *P<0.05; **P<0.01; ***P<0.001.

Figure 8

Figure 8. Allergic responses to OVA/LPS sensitization and challenge in bone marrow chimeric mice

(a) Airway inflammation and (b, c) cytokine production in the OVA/LPS model of asthma. Cytokines are from cultures of whole lungs restimulated ex vivo for 5 days with OVA. Data shown represent mean values ±SEM, and are from one experiment, representative of three. N = 2 to 4 mice/per group. *P<0.05; **P<0.01; ***P<0.001

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