A clustergram showing K=13 for samples spanning the macrophage polarization spectrum and RF-barcodes characteristic of each polarization state.

The clustering pattern was obtained by using M3C, a widely used agnostic clustering method based on Monte Carlo referencing that uses stability selection to estimate the number of clusters (John et al., 2020). (A) The robustness metrics, Relative Cluster Stability Index (RCSI) spiked with a significant p-value (B) at K =13. (C) Cluster-member associations in the consensus matrix for K=13. Clusters with a single member are starred. Clusters 2 and 12 are singletons. (D) RF barcodes which are characteristic of the thirteen different clusters. Each barcode is a heatmap of the differential expression profile of the identified transcription factors (as compared to the unstimulated Mo state) in each cluster. The length of the barcode (which is the number of RFs in each cluster) is indicated within parentheses. Barcodes of members within a cluster resemble each other extensively and are shown together.

Switching factors that guide M1 to M2 destination.

(A) A heatmap of the genes in the barcode that exhibits a reversal in the fold change pattern between M1 (C11) and M2 (C3). RFs that are selected for experimental validation are starred. (B) A regulatory core for the M1 cluster (C11). The core network (nodes: 2028; interactions: 2145) that contains the top perturbed regulatory interactions common across C11 members (sLPS, sLPS+IC and TPP) is shown. Larger triangle nodes represent RFs identified to show switching behaviour; smaller triangles represent RFs present in the C11 barcodes, and circles represent genes common to all C11 members.The nodes are colored based on the fold change (color scale is shown as an inset).

siRNA knockdown of NFE2L2, CEBPB and BCL3 in LPS infected THP-1 derived macrophage cell line attenuates macrophages polarizing to C11 state (M1).

(A) Schema describing the experimental design for testing the role of the 3-gene set NCB, predicted to be a switching factor combination between M1 and M2 destinations. The LPS and IL4 treatment conditions are included to verify the experimental setup. The non-targeting siRNA is used as a negative control while siSTAT4 is used as a positive control. siNCB is a knock-down of the 3-gene set (NCB) which is evaluated in this study. (B) Gene expression changes of selected RFs and known markers of M1 (IL1B) and M2 (IL10) in THP1 cell lines upon LPS and IL4 treatment (for 72 hr) respectively. Color coding scheme is similar to that in (A). THP1 monocytes were treated with PMA (20ng/ml) for 16h. Post 24h, LPS (100ng/ml) and IL-4(1000IU) treatment was given for 72 hr. Two-way ANOVA ( P > 0.05 :ns, P < 0.05:*, P < 0.01:**, P < 0.001:*** ; N=3) is used for estimating significance in gene expression fold change. (C) Gene expression changes in the M1 markers upon siRNA knockdown of STAT4 (positive control). THP1 monocytes were treated with PMA (20ng/ml) for 16h. STAT4 specific siRNA was transfected. Post 8h, LPS (100ng/ml) treatment was given for 72h. Gene expression changes in M1 markers were quantified (Two-way ANOVA; P > 0.05 :ns, P < 0.05:*, P < 0.01:**, P < 0.001:*** ; N=3; NT: non-targeting siRNA) (D) Gene expression changes in the M1 and M2 markers upon siRNA knockdown of the NCB set. THP1 monocytes were treated with PMA (20ng/ml) for 16h. NCB (NFE2L2, CEBPB , BCL3) specific siRNA was transfected. Post 8h, LPS (100ng/ml) treatment was given for 72h. Gene expression changes of M1 and M2 markers were quantified (Two-way ANOVA; P > 0.05 :ns, P < 0.05:*, P < 0.01:**, P < 0.001:*** ; N=3; NT: non-targeting siRNA)

siRNA knockdown of NCB (NFE2L2, CEBPB and BCL3) in S.aureus infected macrophage increases infectivity.

(A) Schema describing the experimental design. (B) Effect of the NCB set knockdown in the S. aureus infection model. siRNA knockdown of NFE2L2, CEBPB and BCL3 in THP-1 cells infected with S. aureus leads to a significant decrease in the gene expression of the M1 markers (CXCL2, IL1B, iNOS and SOCS3) and an increase in the expression of the M2 markers (TGFβ and IL10). Color coding is based on the schema shown in (A). THP1 monocytes were treated with PMA (20ng/ml) for 16hrs. NFE2L2, CEBPB and BCL3 specific siRNAs were transfected. Post 24hrs, S.aureus infection at 1:10 MOI was given for 24hrs. Gene expression levels of M1 and M2 markers were assessed. (Two-way ANOVA; P > 0.05 :ns, P < 0.05:*, P < 0.01:**, P < 0.001:*** ; N=3; NT: non-targeting siRNA). (C) In vitro S.aureus CFU assay. THP-1 cells were differentiated to macrophages with PMA for 16 hrs. Post 24 hrs, siRNA specific for NFE2L2, BCL3 and CEBPB were transfected. S.aureus infection (MOI 10) was given for 2 hrs prior to 2 hrs gentamicin treatment. CFU assay was performed at 0 hr and 24 hrs. (Two-way ANOVA; P > 0.05:ns, P < 0.05:*, P < 0.01:**, P < 0.001:*** ; N=3; NCB : NFE2L2, CEBPB and BCL3). CFU-Colony Forming Units.