Metabolic heterogeneity of tissue-resident macrophages in homeostasis and during helminth infection

Tissue-resident macrophage populations constitute a mosaic of phenotypes, yet how their metabolic states link to the range of phenotypes and functions in vivo is still poorly defined. Here, using high-dimensional spectral flow cytometry, we observe distinct metabolic profiles between different organs and functionally link acetyl CoA carboxylase activity to efferocytotic capacity. Additionally, differences in metabolism are evident within populations from a specific site, corresponding to relative stages of macrophage maturity. Immune perturbation with intestinal helminth infection increases alternative activation and metabolic rewiring of monocyte-derived macrophage populations, while resident TIM4+ intestinal macrophages remain immunologically and metabolically hyporesponsive. Similar metabolic signatures in alternatively-activated macrophages are seen from different tissues using additional helminth models, but to different magnitudes, indicating further tissue-specific contributions to metabolic states. Thus, our high-dimensional, flow-based metabolic analyses indicates complex metabolic heterogeneity and dynamics of tissue-resident macrophage populations at homeostasis and during helminth infection.


Supplementary Figure 3
Gating strategies for identifying tissue macrophage populations from the whole brain, lung, large and small intestines, spleen, liver and peritoneal cavity.

Supplementary Figure 5
Heatmap showing relative expression for gMFI, according to the mean, of metabolic targets detected by spectral flow cytometry, compared to mean expression (log base 2) from bulk RNA-seq data generated by Lavin et al 24 , generated using ClustVis 57 .

Reference controls
Reference controls were stained simultaneously in parallel to the corresponding staining step for the given marker.Beads were used, as we found that this reduced the complexity resulting from AF, and improved resolution of macrophage markers.Cells were used only if beads were not suitable for the antibody isotype, or if the brightness on cells was consistently brighter than on the beads.Cells were used to generate references for all intracellular metabolic targets.Single stain references were reused for subsequent experiments up to ~2 months, or after servicing of the cytometer.

Defining AF/unstain and unmixing
The following is a guideline for defining AF, however exact protocol is subject to each experiment/tissue: • Create a gate to exclude debris, and second gate to exclude aggregates (single-cell gate) • Within single cell gate, exclude UV7-high cells (P3 gate) o This removes large proportion of dead/non-immune cells, as seen in Supplementary Fig. o In general, macrophage AF has similar overall spectra as lymphocytes, so it is not necessary to additionally select the "Autofluorescence as tag" toggle In the event of multiple AF populations it is possible to check their resulting complexity: • Aim to gate the most negative population possible within total cells (debris gate -spectrum should be as "flat" as possible) and export as FCS • Set the "debris" gate as the unstained and import AF populations into reference group • "Unmix", define references and select "QC Controls" tag and "similarity matrix" o Click view similarity index to view the complexity between defined AF channels -anything less than 0.98 can be used as a separate tag Note: defining multiple channels will not always improve unmixing, so a certain trial and error is required to determine optimal AF channels and unmixing.

F480
gating for BMDM after days of culture.(b) Polarization of BMDM in response to overnight stimulation with LPS/IFNγ or IL-4.(c) Example traces for Seahorse mitochondrial stress test for BMDM, representative of three experiments.(d) RELMα expression in control or IL-4 stimulated BMDM cultured with DMSO or ACC inhibitor; data points represent biological replicates from individual mice (n=5), representative of 2 independent experiments.(e) Representative scatter-plots for hMDM after 6 days of culture.(f) Example Seahorse traces for hMDM, representative of four experiments with 2-3 donors per experiments.Statistics determined by two-tailed unpaired t-test.****p<0.0001,***p<0.001,**p<0.01,*p<0.05.Source data are provided as a Source Data file.Supplementary Figure 2 (a) Distinct scatter profiles and spectral signatures of mphs, lymphocytes and eosinophils in the murine peritoneal cavity.(b) Representative unmixing of unstained sample for a channel with high similarity to AF (DL405), and a channel with low similarity to AF (APC-Fire810) unmixing with or without AF channels defined in (a).(c) Identification of immune cell AF signatures in stromal tissues using raw worksheet, with the lung as an example, and representative unmixing of unstained samples for BV510 (high AF overlap) with and without AF definition.(d) Representative unmixing results for TIM4-PerCP-eFluor710 staining on liver mphs with and without defining extra AF channel.(e) Example of increasing resolution of single-stained samples by gating out AF before unmixing.
(a) Comparison of raw gMFIs for metabolic markers across tissue macrophages from 1 of 4 independent experiments.(b) PEC samples divided with half stored on ice and the remaining "digested" in RPMI shaking at 37•C for 30min in parallel to digesting tissues, then assessed for metabolic expression and overlap following UMAP using metabolic targets; data points are individual mice (n=4), representative of 2 experiments.(c) Overlay of scatter profiles for prominent clusters in the liver, spleen and PEC and corresponding histograms for metabolic targets in PEC clusters.(d) Histograms identifying interstitial and alveolar macrophage clusters and contour plots showing matching scatter profiles.Source data are provided as a Source Data file.

F480
(a) Gating for UMAP and phenograph analysis of PEC macrophages, as shown in (b) generated using immune (CD11b, F480, MHCII, CD11c, TIM4, CD206, CX 3 CR1, Ly6C, CD301) and metabolic markers.(c) Expression of immune and metabolic markers for identified peritoneal macrophage clusters; corresponding data to Figure 3c.(d) Overlaid histograms and graphed data comparing HPG uptake of TIM4 -and TIM4 + peritoneal macrophages, corresponding data to Figure 3d.(e) Normalized gene expression of CD36 and PPARγ in MHCII Hi and F480 Hi macrophages, taken from ImmGen Gene Skyline data browser.(f) Comparison of CD36 expression between MHCII Hi and F480 Hi macrophages, gated on CD36 + cells, when stained after 20 minutes of incubation at 37•C or kept on ice, data points are individual mice (n=5, mean±SD).Source data are provided as a Source Data file.
(a) Quantified MitoTracker DeepRed and TMRM staining on small intestine macrophages from naïve (n=4) or Hp infected (n=3) mice, one experiment represented (mean±SD).(b) Representative staining and frequencies for alternative activation markers in TIM4 -monocytes/macrophages and TIM4 + macrophages from the small intestine following H. polygyrus infection, representative of, or pooled from, 2 experiments with 3-5 mice per group.(c) Flow plots displaying monocyte waterfall in naïve, infected and cleared mice, and corresponding frequencies of monocytes, intermediate and mature macrophages.(d) Corresponding flow plots for Ly6C and PDL2 expression and frequency of total PDL2 + cells, individual mice shown (n=5 per group), representative of 2 experiments.(e) UMAP and relative metabolic expression in peritoneal macrophages before, during or after infection, individual mice shown and representative of 2 experiments (n=2-5/group, 25-75 th percentile with median, min and max shown).(f) Gating used to identify PDL2 + /RELMα + macrophages, used for Fig. 6h, and gMFI for metabolic targets in each quadrant, individual mice shown (n=5 per group), representative of 2 experiments.Source data are provided as a Source Data file.
2c • Within P3, use 2-D plot with aid of spectrogram to cycle through common peak AF channels to identify if multiple populations are present o Often, in our hands a combination of B3, UV7 or V7 will identify macrophages and eosinophils o Confirm homogeneity of population with spectrogram • Once AF population has been identified, right click gated area and export as FCS • Similarly for unstained, use 2-D plot of P3 but gate most AF low population • Create a new AF channel (Library -> Fluorescent tags -> select laser group -> "Add" -> define and label AF channel) and add tag to experiment setup o Add corresponding reference under "edit"->"groups" • Import exported FCS files to new AF channel and unstain reference • "Unmix"