Fig 2: As a target gene of STAT6, Zeb1 is important in EMT triggered by IL-4. (A) mRNA levels of EMT transcription factors in HCT116 cells treated with 20 ng/ml IL-4 for 24 h. (B) Western blots of the indicated proteins with specific antibodies in HCT116 cells treated with 20 ng/ml IL-4 for 24 h. (C) mRNA levels of Zeb1/2 in HCT116 cells with STAT6-knockdown. (D) Western blots of the indicated proteins with specific antibodies in HCT116 cells transfected with STAT6 siRNA and exposed to 20 ng/ml IL-4 for 24 h. (E) Western blots of the indicated proteins with specific antibodies in HCT116 cells co-transfected with STAT6 siRNA/Zeb1 overexpression plasmid and exposed to 20 ng/ml IL-4 for 48 h. *P<0.05; **P<0.01. STAT6, signal transducer and activator of transcription 6; EMT, epithelial-mesenchymal transition; IL, interleukin; Zeb, zinc finger E-box-binding homeobox; si, small interfering; Ctrl, control; E-cadherin, epithelial cadherin; ZO-1, zonula occludens-1; EV, empty vector.

Fig 3: Working model for convergent signaling of growth factors and cytokines in the induction of arg1 in VL. L. donovani infection induces the production of IL-4, IL-10 and FGF-2 in the spleen. FGFR is activated, as is IGF-IR by a yet to be identified host or parasite ligand. JAK kinases are phosphorylated through the activated cytokine or growth factor receptors, which lead to IRS-1/2, AKT, ERK, and STAT activation. Translocated STAT6, and possibly STAT3 lead to the transcriptional activation of arginase, which generates polyamines from arginine and leads to parasite growth. These transcription factors also contribute to the polarization macrophages toward an M2 phenotype, which is more permissive to L. donovani survival and growth. The collective effect of AKT, ERK, and STAT3 activation, and the generation of polyamines, are likely to lead to growth, proliferation and survival of arginase expressing cells, but this needs experimental confirmation in VL. Solid lines indicate known mechanistic interactions; dashed lines represent suppositional interactions. Only key shared signaling proteins are included in the model.

Fig 4: Inhibiting Hedgehog signaling upregulates an inflammatory gene expression signature (a) Heat map of the gene signature of macrophages derived from RAW 264.7 cells treated with M-Csf (M0 state), M-Csf and IL-4 (M2 macrophages), M-Csf, IL-4, and DMSO (M2 and vehicle control), and M-Csf, IL-4, and 20 µM GANT61 (M2 inhibited for Hh signaling). GANT61-treated macrophages demonstrate clear differences from vehicle controls. Log2-transformed RPKM-normalized intensities were used for Z-normalization with color indicating above (in red) or below (in blue) average. (b) GANT61-treated macrophages demonstrate similarity to Stat6 (-/-) macrophages, although Stat6 (-/-) and Stat6 (WT) show fewer differences among these genes. (c) Gene set enrichment analysis of RNAseq data depicts that the molecular gene signature of M2 macrophages is characteristically immunosuppressive. Inhibition of Hh/Gli signaling reveals an overlap with inflammatory signature in bacterial infections and antigen presentation by dendritic cells. (d) Heatmap showing expression level of M1 and M2 macrophage genes in normal (n = 114), HER2 Positive (n = 37), Luminal (n = 566), and triple negative breast cancer [TNBC] (n = 116) samples from TCGA breast invasive carcinoma [BRCA] dataset. Transcript per million (TPM) value for each sample is obtained by multiplying RSEM scaled_estimate by 106.The significance of differential expression is estimated via student’s T-test.

Fig 5: The mechanism by which S27 peptide caused anti-inflammatory modulation to PBMMs. Upon dissemination, secreted S27 interact with IL-4/IL-13R complex causing and early phosphorylation of STAT6. Macrophages will polarize into M2-like phenotype and modulation in iron handling proteins that favors the release of iron into the milieu of mucormycosis that support further persistence and dissemination.