Allergen-Induced C5a/C5aR1 Axis Activation in Pulmonary CD11b+ cDCs Promotes Pulmonary Tolerance through Downregulation of CD40

Abstract

Activation of the C5/C5a/C5a receptor 1 (C5aR1) axis during allergen sensitization protects from maladaptive T cell activation. To explore the underlying regulatory mechanisms, we analyzed the impact of C5aR1 activation on pulmonary CD11b⁺ conventional dendritic cells (cDCs) in the context of house-dust-mite (HDM) exposure. BALB/c mice were intratracheally immunized with an HDM/ovalbumin (OVA) mixture. After 24 h, we detected two CD11b⁺ cDC populations that could be distinguished on the basis of C5aR1 expression. C5aR1^- but not C5aR1⁺ cDCs strongly induced T cell proliferation of OVA-reactive transgenic CD4⁺ T cells after re-exposure to antigen in vitro. C5aR1^- cDCs expressed higher levels of MHC-II and CD40 than their C5aR1⁺ counterparts, which correlated directly with a higher frequency of interactions with cognate CD4⁺ T cells. Priming of OVA-specific T cells by C5aR1⁺ cDCs could be markedly increased by in vitro blockade of C5aR1 and this was associated with increased CD40 expression. Simultaneous blockade of C5aR1 and CD40L on C5aR1⁺ cDCs decreased T cell proliferation. Finally, pulsing with OVA-induced C5 production and its cleavage into C5a by both populations of CD11b⁺ cDCs. Thus, we propose a model in which allergen-induced autocrine C5a generation and subsequent C5aR1 activation in pulmonary CD11b⁺ cDCs promotes tolerance towards aeroallergens through downregulation of CD40.

Keywords: C5a receptor; CD40; allergic asthma; complement; conventional dendritic cell; house-dust-mite.

Figure 1

Pulmonary CD11b⁺ cDCs are a heterogeneous cDC population. (A) Gating strategy to identify pulmonary CD11b⁺ cDCs in WT naïve and allergen-exposed BALB/c mice. CD11b⁺ cDCs were identified as SiglecF⁻ lin⁻ CD11C⁺ MHC-II^hi CD103⁻ CD11b⁺ CD64⁻ cells. (B) Histograms showing the expression of C5aR1 in pulmonary CD11b⁺ cDCs from both naive mice (left panel) and animals exposed 1× i.t. to HDM/OVA (right panel). The data shown in the histograms are representative of 10 mice. (C) Frequencies of pulmonary CD11b⁺ C5aR1⁻ and CD11b⁺ C5aR1⁺ cDCs in naïve (left panel) and mice immunized 1× i.t. with HDM/OVA (right panel). Shown are the frequencies of the two subsets within total lung cells as the mean ± standard error of the mean (SEM), n = 10. Differences between groups were determined by unpaired t-test; **** p < 0.0001. (D,E) Dot plots showing the expression of CD301, CD24 and CD209 in pulmonary CD11b⁺ cDCs from (D) naive or (E) mice exposed 1× i.t. to HDM/OVA. The histograms show the frequency of C5aR1⁺ cells within the CD11b⁺ cDC population that were either CD301, CD24 or CD209 positive or negative. (F) Quantitative evaluation of the frequency of C5aR1⁺ or C5aR1⁻ cells within the subsets of CD301, CD24 or CD209 positive or negative cells. The gray histograms represent Fluorescence minus one (FMO) controls.

Figure 2

Proliferation and differentiation of OVA-transgenic CD4⁺ T cells in response to co-culture with OVA-pulsed C5aR1⁺ or C5aR1⁻ cDCs. WT BALB/c mice were treated once with a combination of HDM/OVA (100 μg/40 μg) i.t. C5aR1⁺ and C5aR1⁻ CD11b⁺ cDCs from WT mice were FACS-purified from digested lung tissue 24 h after immunization. Both cell populations were co-cultured with CFSE-labeled OVA-specific TCR tg CD4⁺ T cells in the presence of OVA (10 μΜ) and GM-CSF (20 ng/mL) for four days. (A) Histogram showing the CFSE signal in OVA-specific TCR Tg CD4⁺ T cells after four days of co-culture with either C5aR1⁺ or C5aR1⁻ cDCs. The grey histogram shows the signal obtained from T cell directly after CFSE labeling. (B) Frequency of proliferated OVA-specific TCR tg CD4⁺ T cells, Data shown are the mean ± SEM, n = 15 per group. (C) On day 4, the differentiation of T cells was evaluated by intra-cellular cytokine staining. Shown is the percentage of proliferated CD4⁺ T cells expressing IL-13, IL-17A, IFN-γ or FOXP3 as the mean ± SEM, n = 3–5 per group. Differences between groups were assessed by unpaired t-test; * indicates significant differences between T cell co-cultures of C5aR1⁻ vs. C5aR1⁺ cDCs; * p < 0.05, *** p < 0.001, **** p < 0.0001.

Figure 3

Association of C5aR1 expression on CD11b⁺ cDCs with MHC-II, CD40, CD80, CD86 and OX40L in naïve and HDM/OVA-immunized mice. (A) Costimulatory molecule expression in cDCs from either naïve WT mice (left panel) or WT mice treated once i.t. with a mixture of HDM/OVA (100 μg/40 μg) (right panel). Twenty-four hours after the in vivo treatment, the expression of MHC-II and the co-stimulatory molecules CD40, CD80, CD86, and OX40L was evaluated by measuring 10,000 events for each of the two subpopulations. (B) Comparison of MHC-II and costimulatory expression levels before and after treatment with HDM/OVA (100 μg/40 μg) in C5aR1⁻ cDCs (left panel) and C5aR1⁺ cDCs (right panel). Shown is the ΔMFI of the expression levels of the examined molecules by the two subsets as mean ± SEM; the ΔMFI is defined as the mean fluorescence intensity of the signal normalized to the FMO control; n = 3–6 per group; differences between groups were evaluated by unpaired t-test, * p < 0.05; ** p < 0.01; **** p < 0.0001.

Figure 4

Monitoring of the interaction between OVA-specific TCR tg CD4⁺ T cells and C5aR1⁻ or C5aR1⁺ cDCs. WT mice were treated once with a combination of HDM/OVA (100 μg/40 μg) i.t. C5aR1⁺ and C5aR1⁻ CD11b⁺ cDCs from WT mice were FACS-purified from lung tissue 24 h after allergen exposure. The cDCs were pulsed with the OVA (10 μΜ) and GM-CSF (20 ng/mL) overnight. The following day, they were labeled with PKH26 and transferred in a channel slide together with CFSE-labeled OVA-specific TCR tg CD4⁺ T cells. Using an FV1000 confocal microscope, the interactions between cDCs and T cells were tracked for 300 min (one picture/minute). To visualize the interactions between the two cell populations, the Imaris™ coloc tool was used. It operates simultaneously on two channels (PE and FITC) and measures the degree of overlap between the two channels. (A) Intensity histogram of the PE/FITC channels, which reflects the distribution of voxel pair intensities occurring in the two selected channels (a voxel is a unit of graphic information that defines a point in three-dimensional space). The range of intensity pairs considered as colocalized can be defined on the histogram as channel thresholds, marked with the two yellow lines. The voxel numbers in C5aR1⁻ cDCs (left) were higher than in C5aR1⁺ cDCs (right). (B) Number of voxels per minute monitored for a time period of 300 min using either C5aR1⁻ cDCs (blue) or C5aR1⁺ cDCs (red), n = 4 per group. (C) Quantitative evaluation of the curves shown in (B). The area under the curve (AUC) was determined for evaluation. Data shown are the mean ± SEM; n = 4 per group; differences between groups were evaluated by unpaired t-test, * p < 0.05.

Figure 5

CCR7 expression in pulmonary C5aR1⁺ and C5aR1⁻ cDCs (A) Histogram showing the expression of CCR7 in C5aR1⁺ and C5aR1⁻ cDCs FACS-sorted 24 h after i.t. HDM/OVA immunization (100 μg/40 μg). We determined 10,000 events for each of the two subpopulations. The gray histogram represents the FMO control. (B) Quantitative assessment of CCR7 expression in C5aR1⁻ and C5aR1⁺ cDCs. Shown is the ΔMFI for CCR7 within the two subsets. The ΔMFI is defined as the mean fluorescence intensity of the signal normalized to the FMO control. Data shown are the mean ± SEM, n = 6 per group, Differences between groups were assessed by unpaired t-test, * p < 0.05.

Figure 6

In vitro targeting of C5aR1 strongly enhances the potency of C5aR1⁺ cDCs to promote CD4⁺ T cell proliferation through upregulation of CD40. WT mice were treated once with a combination of HDM/OVA (100 μg/40 μg) i.t. C5aR1⁺ and C5aR1⁻ CD11b⁺ cDCs from WT mice were FACS-purified from lung tissue 24 h after the treatment. C5aR1⁺ cDCs were either treated with 5 μg/mL of the C5aR1-specific neutralizing mAb 20/70 or an appropriate isotype control. (A) cDCs were co-cultured with CFSE-labeled OVA-specific TCR tg CD4⁺ T cells in the presence of OVA (10 μΜ) and GM-CSF (20 ng/mL) for four days. Left panel; histogram detailing the T proliferation in the different treatment groups; the grey histogram shows the signal obtained from T cell directly after CFSE labeling; right panel: quantification of T cell proliferation in the different treatment groups. Values shown are the mean ± SEM; n = 12. Data were analyzed by ANOVA, followed by Tukey’s posthoc test; * indicates significant differences between the different treatment groups, **** p < 0.0001. (B,C) Evaluation of CD40 (B) and MHC-II (C) expression 18 h after in vitro targeting C5aR1. Left panels: histograms showing the expression of CD40 (B) or MHC-II (C) in the presence or absence of C5aR1 targeting, grey histogram = FMO control; right panels: quantitative assessment of CD40 or MHC-II expression in the presence or absence of C5aR1 targeting. Shown is the expression of CD40 or MHC-II (as ΔMFI) in the two treatments groups as mean ± SEM; the ΔMFI is defined as the mean fluorescence intensity of the signal normalized to the FMO control, n = 6–9 per group. Data were analyzed with unpaired t-test; * indicates significance between the two treatment groups, ** p < 0.001. (D) Assessment of CD40L targeting on T cell proliferation in C5aR1⁺ cDCs, in which C5aR1 signaling was blocked. Left panel: histogram showing the impact of C5aR1 blockade vs. C5aR1 and CD40/CD40L blockade on T cell proliferation; the grey histogram shows the signal obtained from T cell directly after CFSE labeling. The graph on the right side shows the quantitative evaluation of the different treatment groups. Values shown are the mean ± SEM; n = 15 per group. Data were compared by ANOVA followed by Tukey posthoc test. * indicates significant differences between the treatment groups; **** p < 0.0001. (E) Impact of C5aR1 targeting on T cell proliferation when the antigen concentration is limited. WT mice were treated once with HDM (100 μg) i.t. C5aR1⁺ cDCs from WT mice were FACS-purified from lung tissue 24 h after the treatment. C5aR1⁺ cDCs were co-cultured with CFSE-labeled OVA-specific TCR tg CD4⁺ T cells in the presence of different concentrations of OVA^323–339 peptide (5 μg/mL, 500 ng/mL, 50 ng/mL, 20 ng/mL and 5 ng/mL) and GM-CSF (20 ng/mL). Left panel: frequency of proliferated T cells in response to different concentrations of OVA^323−339 peptide as mean ± SEM, n = 3–9. Data were analyzed by ANOVA, followed by Tukey post-hoc test; * indicates significant differences between the different treatment groups, **** p < 0.0001; right panel: impact of C5aR1 targeting on C5aR1⁺ cDC-driven T cell proliferation in the presence of low OVA^323–339 peptide concentration, n = 6–10. Data were analyzed by unpaired t test; * indicates significant differences between the two treatment groups; *** p < 0.001.

Figure 7

The effect of MHC-II targeting on C5aR1⁻ cDC-driven T cell proliferation in response to CD40L neutralization. (A) Histogram (left) showing the impact of MHC-II (10 pg/mL) blockade on T cell proliferation in C5aR1⁻ cells in the presence or absence of CD40L blockade; the grey histogram shows the signal obtained from T cell directly after CFSE labeling. The graph on the right-hand side shows the quantitative evaluation of MHC-II ± CD40L blockade. Values shown are the mean ± SEM; n = 6–8 per group. Data were analyzed by ANOVA, followed by Tukey post-hoc test; * indicates significant differences between the treatment groups; *** p < 0.001 and **** p < 0.0001. (B) Impact of CD40L targeting on T cell proliferation when the antigen concentration is limited. WT mice were treated once with HDM (100 μg) i.t. C5aR1⁻ cDCs from WT mice were FACS-purified from lung tissue 24 h after the treatment. C5aR1⁻ cDCs were co-cultured with CFSE-labeled OVA-specific TCR tg CD4⁺ T cells in the presence of different concentrations of OVA^323–339 peptide (5 μg/mL, 500 ng/mL, 50 ng/mL, 20 ng/mL and 5 ng/mL) and GM-CSF (20 ng/mL). Left panel: frequency of proliferated T cells in response to different concentrations of OVA^323–339 peptide as mean ± SEM, n = 6–7. Data were analyzed by ANOVA, followed by Tukey post-hoc test; * indicates significant differences between the different treatment groups, **** p < 0.0001; right panel: impact of CD40L targeting on C5aR1⁻ cDC-driven T cell proliferation in the presence of low OVA^323–339 peptide concentration, n = 6–7. Data were analyzed by unpaired t test; * indicates significant differences between the two treatment groups; **** p < 0.0001.

Figure 8

Impact of OVA-pulsing and T cell co-culture on C5 production and C5a generation from C5aR1⁺ and C5aR1⁻ cDCs. (A,F) Histograms showing C5 production (A) or C5a generation (F) in C5aR1⁺ or C5aR1⁻ cDC subsets directly after FACS purification on day 0, after OVA-pulsing on day 1 or after addition of OVA-tg CD4⁺ T cells on day 2; grey histogram = FMO control. (B,G) Frequency of C5-producing (B) or C5a-generating (G) C5aR1⁺ (left panel) or C5aR1⁻ cDCs (right panel). (C,H) Comparison of the frequencies of C5-producing (C) or C5a-generating (H) C5aR1⁺ and C5aR1⁻ cDCs on days 0, 1 and 2. (D,I) Quantitative evaluation of C5 production (D) or C5a generation (I) in C5aR1⁺ (left panel) or C5aR1⁻ (right panel) cDCs on days 0, 1 and 2. (E,J) Comparison of C5 production (E) or C5a-generation (J) in C5aR1⁻ or C5aR1⁺ cDCs on days 0, 1 and 2. The data shown in D, E, I and J show the ΔMFI of C5 or C5a expression by the two cDC subsets; the ΔMFI is defined as the mean fluorescence intensity of the signal normalized to the FMO control. Data shown in B–E and G–J are the mean ± SEM, n = 4–6 per group; data in B, D, G and I were analyzed by ANOVA followed by Tukey’s post-hoc test; * indicates significant differences between C5aR1⁺ and C5aR1⁻ cDCs on day 0 vs. days 1 and 2; ** p < 0.01, *** p < 0.001, **** p < 0.0001; data in C, E, H and J were compared by unpaired t-test; * indicates significant differences between C5aR1⁺ and C5aR1⁻ cDCs on days 0, 1 or 2; ** p < 0.01, **** p < 0.0001. (K) Impact of T cells on OVA-driven C5a production C5aR1⁺ (left panel) or C5aR1⁻ (right panel) cDCs from allergen-exposed mice. Shown is the intracellular expression of C5a in OVA-pulsed cDCs that were co-cultured with or without OVA-specific TCR tg CD4⁺ T cells as ΔMFI of C5a expression by the two cDC subsets. Data shown are the mean ± SEM. n = 9. They were analyzed by unpaired t-test. * p < 0.05.

Figure 8

Impact of OVA-pulsing and T cell co-culture on C5 production and C5a generation from C5aR1⁺ and C5aR1⁻ cDCs. (A,F) Histograms showing C5 production (A) or C5a generation (F) in C5aR1⁺ or C5aR1⁻ cDC subsets directly after FACS purification on day 0, after OVA-pulsing on day 1 or after addition of OVA-tg CD4⁺ T cells on day 2; grey histogram = FMO control. (B,G) Frequency of C5-producing (B) or C5a-generating (G) C5aR1⁺ (left panel) or C5aR1⁻ cDCs (right panel). (C,H) Comparison of the frequencies of C5-producing (C) or C5a-generating (H) C5aR1⁺ and C5aR1⁻ cDCs on days 0, 1 and 2. (D,I) Quantitative evaluation of C5 production (D) or C5a generation (I) in C5aR1⁺ (left panel) or C5aR1⁻ (right panel) cDCs on days 0, 1 and 2. (E,J) Comparison of C5 production (E) or C5a-generation (J) in C5aR1⁻ or C5aR1⁺ cDCs on days 0, 1 and 2. The data shown in D, E, I and J show the ΔMFI of C5 or C5a expression by the two cDC subsets; the ΔMFI is defined as the mean fluorescence intensity of the signal normalized to the FMO control. Data shown in B–E and G–J are the mean ± SEM, n = 4–6 per group; data in B, D, G and I were analyzed by ANOVA followed by Tukey’s post-hoc test; * indicates significant differences between C5aR1⁺ and C5aR1⁻ cDCs on day 0 vs. days 1 and 2; ** p < 0.01, *** p < 0.001, **** p < 0.0001; data in C, E, H and J were compared by unpaired t-test; * indicates significant differences between C5aR1⁺ and C5aR1⁻ cDCs on days 0, 1 or 2; ** p < 0.01, **** p < 0.0001. (K) Impact of T cells on OVA-driven C5a production C5aR1⁺ (left panel) or C5aR1⁻ (right panel) cDCs from allergen-exposed mice. Shown is the intracellular expression of C5a in OVA-pulsed cDCs that were co-cultured with or without OVA-specific TCR tg CD4⁺ T cells as ΔMFI of C5a expression by the two cDC subsets. Data shown are the mean ± SEM. n = 9. They were analyzed by unpaired t-test. * p < 0.05.

Figure 8

Impact of OVA-pulsing and T cell co-culture on C5 production and C5a generation from C5aR1⁺ and C5aR1⁻ cDCs. (A,F) Histograms showing C5 production (A) or C5a generation (F) in C5aR1⁺ or C5aR1⁻ cDC subsets directly after FACS purification on day 0, after OVA-pulsing on day 1 or after addition of OVA-tg CD4⁺ T cells on day 2; grey histogram = FMO control. (B,G) Frequency of C5-producing (B) or C5a-generating (G) C5aR1⁺ (left panel) or C5aR1⁻ cDCs (right panel). (C,H) Comparison of the frequencies of C5-producing (C) or C5a-generating (H) C5aR1⁺ and C5aR1⁻ cDCs on days 0, 1 and 2. (D,I) Quantitative evaluation of C5 production (D) or C5a generation (I) in C5aR1⁺ (left panel) or C5aR1⁻ (right panel) cDCs on days 0, 1 and 2. (E,J) Comparison of C5 production (E) or C5a-generation (J) in C5aR1⁻ or C5aR1⁺ cDCs on days 0, 1 and 2. The data shown in D, E, I and J show the ΔMFI of C5 or C5a expression by the two cDC subsets; the ΔMFI is defined as the mean fluorescence intensity of the signal normalized to the FMO control. Data shown in B–E and G–J are the mean ± SEM, n = 4–6 per group; data in B, D, G and I were analyzed by ANOVA followed by Tukey’s post-hoc test; * indicates significant differences between C5aR1⁺ and C5aR1⁻ cDCs on day 0 vs. days 1 and 2; ** p < 0.01, *** p < 0.001, **** p < 0.0001; data in C, E, H and J were compared by unpaired t-test; * indicates significant differences between C5aR1⁺ and C5aR1⁻ cDCs on days 0, 1 or 2; ** p < 0.01, **** p < 0.0001. (K) Impact of T cells on OVA-driven C5a production C5aR1⁺ (left panel) or C5aR1⁻ (right panel) cDCs from allergen-exposed mice. Shown is the intracellular expression of C5a in OVA-pulsed cDCs that were co-cultured with or without OVA-specific TCR tg CD4⁺ T cells as ΔMFI of C5a expression by the two cDC subsets. Data shown are the mean ± SEM. n = 9. They were analyzed by unpaired t-test. * p < 0.05.

Figure 9

Model detailing the impact of C5a/C5aR1 axis activation in C5aR1⁺ cDCs on the activation of antigen specific CD4⁺ T cells. (A) Low potency of C5aR1⁺ cDCs to drive proliferation of antigen specific CD4⁺ T cells. C5a-mediated activation of C5aR1 suppresses the expression of CD40 in MHC-II^lo C5aR1⁺ cDCs. Under such conditions CD40 is critical for cDC-driven proliferation of antigen-specific T cells. (B) C5aR1-targeting of C5aR1⁺ cDCs increases their potency to drive CD4⁺ T cell proliferation. The lack of C5aR1 signaling results in increased CD40 expression in MHC-II^lo C5aR1⁺ cDCs leading to improved synapse formation and stronger T cell activation.