F4 80 af647

Isolation of Intestinal lamina propria cells was performed by following a method established previously58 (link) with slight modifications. Small intestines were washed with three times with HBSS (Ca/Mg-free), and fat and Peyer's patches were removed. Small intestines were then opened longitudinally, cut into 1-cm pieces, and incubated in HBSS containing 5 uM EDTA+5%FBS+1μM DTT. Tissue was then digested 0.14 Wünsch U ml⁻¹ Liberase (Sigma) for 30 mins at 37 °C on a rotor. The digested cell suspension was then passed through 100 μm cell strainers. Isolated intestinal cells were stained with CD45 Percpcy5.5 (0.35 μl per 100 μl; #45–0451–82, eBioscience, San Diego, CA) CX3CR1 PE-TexasRed (1.5 μl per 100 μl; #149013, Biolegend, San Diego, CA) F4/80 AF647 (8 μl per 100 μl; #MCA497A647, Bio-Rad, Hercules, CA), CD11b eflour605 (2 μl per 100 μl; #83–0112–42, eBioscience) Ly6C PE/cy7 (1 μl per 100 μl; #128018, Biolegend) Ly6G APC/cy7 (0.3 μl per 100 μl; #127624, Biolegend) and were subjected to flowcytometric sorting to purify intestinal macrophages using FACS Aria machine (BD).

Brain and alveolar lavage cell suspensions, were incubated with 1% Human IgG1 Fc block (R&D systems) for 30 min. Samples were centrifuged, supernatant removed and cells resuspended in a combination of mouse anti‐human CD11b:Pacific Blue (Biolegend) and mouse anti‐pig CD45:AF647 antibodies (BioRad) for microglial isolation, or mouse anti‐pig CD163:FITC (Biorad) and F4/80:AF647 (mouse anti‐pig ADGRE1; ROS‐4E12‐3E6) (Waddell et al., 2018 (link)) antibodies for alveolar macrophage isolation for 30 min incubation at room temperature. Samples were centrifuged, supernatant removed and cells resuspended in FACS buffer. Samples were sorted on a FACSAria III cell sorter. Microglia in brain cell suspensions were identified as CD11b^hiCD45^lo and macrophages identified from the alveolar lavage suspensions as F4/80⁺CD163^lo. Sorted cells were collected directly into TRIzol reagent (Invitrogen) for immediate RNA isolation.