Absence of c-Maf and IL-10 enables type I IFN enhancement of innate responses to LPS in alveolar macrophages

Abstract

Alveolar macrophages (AMs) are lung-resident myeloid cells and airway sentinels for inhaled pathogens and environmental particles. While AMs can be highly inflammatory in response to respiratory viruses, they do not mount proinflammatory responses to all airborne pathogens. For example, we previously showed that AMs fail to mount a robust proinflammatory response to Mycobacterium tuberculosis. Here, we address this discrepancy by investigating the capacity of murine AMs for direct innate immune sensing, using LPS as a model. Use of LPS-coated fluorescent beads enabled us to distinguish between directly exposed and bystander cells to measure transcriptional responses, by RNA-sequencing after cell sorting, and cytokine responses, by flow cytometry. We find that AMs have decreased proinflammatory responses to low-dose LPS compared to other macrophage types (bone marrow-derived macrophages, peritoneal macrophages), as measured by TNF, IL-6, Ifnb, and Ifit3. The reduced response to low-dose LPS correlates with minimal TLR4 and CD14 surface expression, despite sufficient internal expression of TLR4. We also find that AMs do not produce IL-10 in response to a variety of stimuli due to low expression of the transcription factor c-Maf, while exogenous c-Maf expression restores IL-10 production in AMs. Lastly, we show that lack of IL-10 enables type I IFN enhancement of AM responses to LPS. Overall, we demonstrate AMs have a cell-intrinsic hyporesponsiveness to LPS, which makes them uniquely tolerant to low-dose exposure. Regulation of AM innate responses by distinct CD14, c-Maf, and IL-10 expression patterns has important implications for both respiratory infections and environmental airborne exposures.

Keywords: IL-10; alveolar macrophages; c-Maf; innate immunity; type I IFN.

Figure 1.

Alveolar macrophages do not mount a proinflammatory response to LPS-conjugated beads in vivo or ex vivo. (A) Schematic representation of LPS-coated beads delivered to mice by nebulization or to BMDMs. Uncoated beads resuspended in PBS (PBS bead) delivered to controls. Cells were isolated and sorted into Bead+ or bystander groups. (B) Gene Set Enrichment Analysis of the top 10 differentially expressed pathways between LPS_Bead BMDMs and LPS_Bead AMs. (C) Scatterplot of log₂ fold-change values for AM LPS_Bead and BMDM LPS_Bead populations. Labeled genes are significantly up-regulated (FDR < 0.05, FC > 2) in only the BMDM LPS_Bead population. (D) Schematic representation of AMs or BMDMs stimulated with LPS-coated beads ex vivo and subsequently analyzed by flow cytometry for intracellular TNF and IL-6. (E) Representative dot plots of the conditions for BMDMs and AMs. (F) Intracellular cytokine staining of TNF and IL-6 for LPS_bead, PBS_bead, or untreated BMDMs and AMs. Data are representative of 3 independent experiments (E) or compiled from 2 independent experiments (B, C) or 3 independent experiments (F). Technical replicates within each experiment represented by unique shapes. ****P < 0.0001; ns, not significant—by 2-way ANOVA with Šídák’s multiple comparison test.

Figure 2.

Alveolar macrophages have a high LPS-specific activation threshold. (A) TNF (left) and IL-6 (right) for AMs (pink, technical duplicate) and BMDMs (black, technical triplicate) either untreated or stimulated with 0.1, 1, or 10 ng/mL of LPS for 20 hours. (B) TNF (left) and IL-6 (right) of BMDMs and AMs stimulated with Pam3Cys (0 to 1,000 ng/mL) for 20 hours. (C) TNF (left) and IL-6 (right) of BMDMs and AMs stimulated with R848 (0 to 100 µg/mL) for 20 hours. (D) Ifnb and Ifit3 gene expression for 0 to 10 ng/mL LPS for BMDMs (black) and AMs (pink) after 4 hours. (E) RT-qPCR of Ifnb (left) and Ifit3 (right) expression relative to Gapdh for 2ʹ3ʹ-cGAMP (0 to 10 µg/mL) after 4 hours. AMs (pink), technical duplicate; BMDMs (black), technical triplicate. (F) phospho-STAT1 intracellular staining MFI of BMDMs (black, technical triplicate) or AMs (pink, technical duplicate) after 20 hours of LPS (1 ng/mL) or fresh media (untreated). Data are representative of 3 independent experiments (A, B, F), 2 independent experiments (C, E) or compiled from 3 independent experiments (D). Technical replicates within each experiment represented by unique shapes. (A–C) *P < 0.05; ** P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant—by 2-way ANOVA with Šídák’s multiple comparison test.

Figure 3.

Alveolar macrophages have low surface expression of TLR4 and CD14 compared to other cell types. (A) Gene expression (log₂ CPM) of AMs (pink) and BMDMs (black) collected in Fig. 1A. (B) TLR4 MFI of AMs (pink, graphed independently to the right) and BMDMs (black) after 0, 1, and 4 hours of LPS (1 ng/mL) stimulation ex vivo. (C) CD14 MFI under the same conditions as (B). (D) Surface and internal TLR4 expression of BMDMs and AMs after no treatment (blue), 1 hour (red), or 4 hours (orange) post–LPS stimulation (1 ng/mL). Gates set based on cell-specific FMOs. (E) Internal TLR4 MFI for both BMDMs (black) and AMs (pink). (F) Frequency of Q3⁺ (internal TLR4⁺) BMDMs and AMs. (G) Surface and internal CD14 expression of AMs and BMDMs before and after stimulation with 4-hour LPS (1 ng/mL). Gates set based on cell-specific FMOs. (H) Internal CD14 MFI for both BMDMs (black) and AMs (pink). (I) Frequency of Q3⁺ (internal CD14⁺) BMDMs and AMs. Data representative of 3 independent experiments (D, G) or compiled from 3 independent experiments (B, C, E, F, H, I). (A–I) *P < 0.05; ****P < 0.001; ns, not significant—by 2-way ANOVA with Šídák’s multiple comparison test.

Figure 4.

Alveolar macrophages do not produce IL-10 in response to multiple stimuli due to low c-Maf expression. (A) IL-10 for AMs (pink) and BMDMs (black) untreated or stimulated with 0.1, 1, or 10 ng/mL of LPS for 20 hours. (B) IL-10 for AMs and BMDMs after 20 hours of Pam3Cys (0 to 1,000 ng/mL). (C) IL-10 for AMs and BMDMs after 20 hours of R848 (0 to 100 µg/mL). (D) IL-10 for AMs and BMDMs after 20 hours of CpG (0 to 10 µM). (E) Expression of IL-10 promoter–associated genes (log₂ CPM) from bulk RNA-sequencing of untreated AMs (left) and BMDMs (right) after 4 hours using data from Fig. 1A. *Adj. P-value < 0.01. (F) c-Maf MFI for BMDMs (black) or AMs (pink) after LPS (10 ng/mL) stimulation or untreated. (G) Maf gene expression relative to Gapdh for mexAMs without polybrene or lentivirus (untreated), with polybrene only (+PB, no LV) , control lentivirus (Empty-LV), or Maf-expressing lentivirus (Maf-LV). (H) Il10 gene expression relative to Gapdh for mexAMs under the same conditions for (G). (I) Il10 gene expression relative to Gapdh for mexAMs transduced with Empty or Maf LV, then stimulated with LPS (10 ng/mL) and IFNβ (100 ng/mL) for 4 hours. (J) IL-10 for mexAMs transduced with Empty or Maf-LV, then stimulated with LPS (10 ng/mL) and IFNβ (100 ng/mL) for 24 hours. (K) ScRNAseq data acquired from CZ CELLxGENE Discover from healthy human lung tissue. Data shown are raw counts normalized to counts per million and log transformed. Data compiled from 3 independent experiments (A–D, F–H) or 2 independent experiments (I, J). (A–D, F, J) *P < 0.05, **P < 0.01, *** P < 0.001, ****P < 0.0001 by 2-way ANOVA with Šídák’s multiple comparison test. (G, H) *P < 0.05 by one-way ANOVA with Tukey’s multiple comparisons test. (I) P-value reported by Mann-Whitney test.

Figure 5.

IFNβ enhances alveolar macrophage TNF and IL-6 response to low-dose LPS in the absence of IL-10. (A) TNF, IL-6, and IL-10 of BMDMs stimulated with LPS (1 ng/mL) with 0.01 to 100 ng/mL of IFNβ. Data shown are mean with SD. (B) AMs or (C) mexAMs stimulated with LPS (1 ng/mL), IFNβ (10 ng/mL), and/or IL-10 (50 µg/mL) for 20 hours followed by ELISA for IL-6 (left) and TNF (right). (D) BMDMs stimulated with LPS (1 ng/mL), IFNβ (10 ng/mL), anti-IL-10R (10 µg/mL), and/or isotype control (Rat IgG1κ, 10 μg/mL). Data are representative of 3 independent experiments (A–D). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant. (A) One-way ANOVA with Šídák’s multiple comparison test, compared to LPS-only condition. (B–D) One-way ANOVA with Tukey’s multiple comparison test.