Innate immune stimuli modulate bone marrow-derived dendritic cell production in vitro by toll-like receptor-dependent and -independent mechanisms

Abstract

Haematopoiesis is crucial for immunity because it results in the production of leucocytes. Bacterial and viral infections alter leucocyte production by promoting granulopoiesis or lymphopoiesis. Recent studies suggest that changes in leucocyte production may be caused by the effects of inflammatory responses on the differentiation of haematopoietic progenitors in the bone marrow. We investigated the mechanisms through which infection regulates the formation of bone marrow-derived dendritic cells (BMDCs) in vitro. We mimicked infection by stimulating developing cells with molecules associated with bacteria and viruses and with inactivated influenza viruses. We showed that toll-like receptor (TLR) ligands act as modulators of haematopoiesis, and that signalling through different TLRs results in differing effects on the production of BMDCs. We demonstrated that ligands for TLR3 and influenza viruses reduce the production of BMDCs, resulting in increased neutrophil numbers, and that ligands for TLR4 and TLR9 drive the production of plasmacytoid dendritic cells. Furthermore, there are distinct signalling mechanisms involved in these effects. Signalling pathways triggered by TLR4 and TLR9 involve MyD88 and are partially mediated by the cytokine tumour necrosis factor-α (TNF-α). Mechanisms activated by TLR3 were Tir-domain-containing adaptor-inducing interferon dependent. Haematopoietic modulation induced by inactivated influenza viruses was associated with the activation of an antiviral pathway mediated by type-1 interferons.

Figure 1

Influenza viruses and bacterial and viral ligands reduce the production of bone marrow-derived dendritic cells (DCs). BALB/c bone marrow cells (5 × 10⁵ cells) were cultured with granulocyte–macrophage colony-stimulating factor (GM-CSF), with or without (a) Jap, X31 or PR8 or (b) Poly I, Poly I:C, lipopolysaccharide (LPS) or cytosine–phosphate–guanosine (CpG) oligodeoxynucleotide (ODN) for 6 days. Surface markers were analysed by flow cytometry. The results are based on data for 10 000 gated events. The numbers shown in the density plots indicate the percentage of cells falling into two defined regions representing CD11c⁺ major histocompatibility complex class II (MHCII)⁺ cells (upper right) or CD11c^lo MHCII^lo cells (lower left). The data shown are the representative results of three similar experiments. (c) Cell numbers were determined by counting on a Neubauer haemocytometer. The counts represent the mean values of triplicate wells for three similar experiments. *P < 0·05, **P < 0·001 and ***P < 0·0001, determined using the Student's t-test, with respect to Nil.

Figure 2

Influenza viruses and bacterial and viral agents induce the production of different cell types. BALB/c bone marrow cells (5 × 10⁵ cells) were cultured for 6 days with granulocyte–macrophage colony-stimulating factor (GM-CSF) in the presence or absence of (a) Jap, X31, PR8, or the toll-like receptor 3 (TLR3) ligands Poly I or Poly I:C and (b) lipopolysaccharide (LPS) or cytosine–phosphate–guanosine (CpG) oligodeoxynucleotide (ODN). Cells were spun and fixed onto slides and then stained with haematoxylin and eosin. Photographs were taken using a Nikon light microscope at 200× magnification. Data shown are representative of three experiments. (c) Cell numbers were assessed under a Nikon light microscope at 400× magnification. Data shown represent the average cell counts from five fields of view for three similar experiments. **P < 0·001 and ***P < 0·0001, determined using the Student's t-test with respect to Nil. (d) BALB/c bone marrow cells (5 × 10⁵ cells) were cultured for 6 days with GM-CSF in the presence or absence of LPS or CpG ODN. CD19 expression was analysed by flow cytometry. Histograms represent unstimulated cultures (shaded histograms) and stimulated cultures (white histograms). Data shown are representative of two experiments. (e) CD11c, major histocompatibility complex class II (MHCII), Gr1 and B220 expression was analysed by flow cytometry. Cells were gated to low expression of CD11c and MHCII and the expression of Gr1 and B220 in this population was assessed. ***P < 0·0001, determined using the Student's t-test with respect to Nil. (f) CD11c, MHCII and PDCA expression was analysed using flow cytometry. Cells were gated to low expression of CD11c and MHCII and the expression of PDCA in this population was assessed. Results are based on 10 000 gated events. The data shown are representative of two similar experiments. **P < 0·001 and ***P < 0·0001, determined using the Student's t-test with respect to Nil.

Figure 3

Effects on bone marrow-derived dendritic cells (BMDC) production are differentially dependent on MyD88 and Tir-domain-containing adaptor-inducing interferon (TRIF). (a) MyD88^+/+ and MyD88^−/− bone marrow cells (5 × 10⁵ cells) were cultured for 6 days with granulocyte–macrophage colony-stimulating factor (GM-CSF) in the presence of lipopolysaccharide (LPS), Jap, X31 or PR8. ***P < 0·0001, determined using the Student's t-test. (b) TRIF^+/+ and TRIF^−/− bone marrow cells (5 × 10⁵) were cultured for 6 days with GM-CSF in the presence or absence of Poly I, Poly I:C, LPS, cytosine–phosphate–guanosine (CpG) oligodeoxynucleotide (ODN), Jap, X31 or PR8. Surface markers were analysed by flow cytometry. The results are based on data for 10 000 gated events. The data shown are representative of two similar experiments. *P < 0·05, **P < 0·001 and ***P < 0·0001, determined using the Student's t-test.

Figure 4

Lipopolysaccharide (LPS) requires toll-like receptor 4 (TLR4) to modulate the production of bone marrow-derived dendritic cells (BMDC). TLR4^+/+ and TLR4^−/− bone marrow cells (5 × 10⁵ cells) were cultured for 6 days with granulocyte–macrophage colony-stimulating factor (GM-CSF) in the presence or absence of Poly I, Poly I:C, LPS or cytosine–phosphate–guanosine (CpG) oligodeoxynucleotide (ODN). Surface markers were analysed by flow cytometry. The results are based on data for 10 000 gated events. The data shown are representative of two similar experiments. ***P < 0·0001, determined using the Student's t-test.

Figure 5

Influenza viruses require type 1 interferons (IFNs) to modulate the production of bone marrow-derived dendritic cells (BMDCs). (a) Type 1 IFN receptor (IFNAR)^+/+ and IFNAR^−/− bone marrow cells (5 × 10⁵ cells) were cultured for 6 days with granulocyte–macrophage colony-stimulating factor (GM-CSF) in the presence or absence of Jap, X31 or PR8. **P < 0·001 and ***P < 0·0001, determined using the Student's t-test. (b) BALB/c bone marrow cells (5 × 10⁵ cells) were cultured for 6 days with GM-CSF in the presence of Jap or recombinant IFN-α. *P < 0·05 and **P < 0·001, determined using the Student's t-test with respect to Nil. (c) BALB/c bone marrow cells (5 × 10⁵ cells) were cultured for 6 days with GM-CSF in the presence of 100 pg/ml of recombinant IFN-α (rIFN-α) or in the presence of rIFN-α plus 200 pg/ml of anti-IFN-α. **P < 0·001, determined using the Student's t-test with respect to rIFN-α. (d) BALB/c bone marrow cells (5 × 10⁵ cells) were cultured for 6 days with GM-CSF in the presence of Jap or in the presence of Jap plus 200 pg/ml of anti-IFN-α. Surface markers were analysed by flow cytometry. The results are based on data for 10 000 gated events. Data shown are representative of two similar experiments. **P < 0·001, determined using the Student's t-test with respect to Jap.

Figure 6

Signalling to modulate changes in the production of bone marrow-derived dendritic cells (BMDCs) in response to lipopolysaccharide (LPS) or cytosine–phosphate–guanosine (CpG) oligodeoxynucleotide (ODN) is partially dependent on tumour necrosis factor-α (TNF-α). BALB/c bone marrow cells (5 × 10⁵ cells) were cultured for 6 days with granulocyte–macrophage colony-stimulating factor (GM-CSF) in the presence or absence of (a) LPS, CpG ODN or recombinant TNF-α. **P < 0·001 and ***P < 0·0001, determined using the Student's t-test with respect to Nil. (b) Recombinant TNF-α in the presence or absence of anti-TNF-α. ***P < 0·0001, determined using the Student's t-test with respect to TNF-α. (c) LPS or CpG ODN in the presence or absence of anti-TNF-α for 6 days. Surface markers were analysed using flow cytometry. The results are based on data for 10 000 gated events. Data shown are representative of two similar experiments. (d) Cell numbers were determined by counting on a Neubauer haemocytometer. The counts represent the mean values of triplicate wells for three similar experiments. **P < 0·001, determined using the Student's t-test with respect to LPS or CpG ODN, as appropriate.