TGFß primes alveolar-like macrophages to induce type I IFN following TLR2 activation.
Alveolar macrophages (AMs) are key mediators of lung function and are potential targets for therapies during respiratory infections. TGFb is an important regulator of AM differentiation and maintenance but, how TGFb directly modulates the innate immune responses of AMs remains unclear. This shortcoming prevents effective targeting of AMs to improve lung function in health and disease. Here we leveraged an optimized ex vivo AM model system, fetal-liver derived alveolar-like macrophages (FLAMs), to dissect the role of TGFb in AMs. Using transcriptional analysis, we first globally defined how TGFb regulates gene expression of resting FLAMs. We found that TGFb maintains the baseline metabolic state of AMs by driving lipid metabolism through oxidative phosphorylation and restricting inflammation. To better understand inflammatory regulation in FLAMs, we next directly tested how TGFb alters the response to TLR2 agonists. While both TGFb (+) and TGFb (-) FLAMs robustly responded to TLR2 agonists, we found an unexpected activation of type I interferon (IFN) responses in FLAMs and primary AMs in a TGFb-dependent manner. Surprisingly, mitochondrial antiviral signaling protein and the interferon regulator factors 3 and 7 were required for IFN production by TLR2 agonists. Together, these data suggest that TGFb modulates AM metabolic networks and innate immune signaling cascades to control inflammatory pathways in AMs. Overall design: FLAMs with and without TGFß were plated in 6-well plates at 1 x 106 cells/well and treated with Pam3 as described for 18 hours. We used the Direct-zol RNA Extraction Kit (Zymo Research, Cat no. R2072) to extract RNA according to the manufacturer's protocol. Quality was assessed by the MSU Genomics Core using an Agilent 4200 TapeStation System. The Illumina Stranded mRNA Library Prep kit (Illumina, Cat no. 20040534) with IDT for Illumina RNA Unique Dual Index adapters was used for library preparation following the manufacturer's recommendations but using half-volume reactions. Qubit™ dsDNA HS (ThermoFischer Scientific, Cat no. Q32851) and Agilent 4200 TapeStation HS DNA1000 assays (Agilent, Cat no. 5067-5584) were used to measure quality and quantity of the generated libraries. The libraries were pooled in equimolar amounts, and the Invitrogen Collibri Quantification qPCR kit (Invitrogen, Cat no. A38524100) was used to quantify the pooled library. The pool was loaded onto 2 lanes of a NovaSeq S4 flow cell, and sequencing was performed in a 2x150 bp paired end format using a NovaSeq 6000 v1.5 100-cycle reagent kit (Illumina, Cat no. 20028316). Base calling was performed with Illumina Real Time Analysis (RTA; Version 3.4.4), and the output of RTA was demultiplexed and converted to the FastQ format with Illumina Bcl2fastq (Version 2.20.0). RNAseq analysis was completed using the MSU High Performance Computing Center (HPCC). FastQC (Version 0.11.7) was used to assess read quality. Bowtie2 (Version 2.4.1) with default settings was used to map reads with the GRCm39 mouse reference genome. Aligned reads counts were assessed using FeatureCounts from the Subread package (Version 2.0.0). Differential gene expression analysis was conducted using the DESeq2 package (Version 1.36.0) in R (Version 4.2.1).