ghted the pathways “Rheumatoid arthritis” and “Cytokine-cytokine receptor interaction” in all models, “Leishmaniasis” in models 2 and three, although “Proteoglycans in cancer”, “Complement and coagulation cascades”, “ECM-receptor interaction”, “Hematopoietic cell lineage”, “Inflammatory bowel disease”, “Legionellosis” and “Salmonella infection” showed a model-specific fashion (Figures S2D ). In contrast, 1,25(OH)2D3 triggered pathways inside a much more diverse way: “Phagosome”, “Staphylococcus aureus infection”, “Tuberculosis”, “Rheumatoid arthritis” and “Leishmaniasis” related with two models, while “Hematopoietic cell lineage”,Frontiers in Immunology | frontiersin.orgDecember 2021 | Volume 12 | ArticleMalmberg et al.Vitamin D Treatment Sequence Is CriticalADGBEHCFIFIGURE 2 | Genes and pathways impacted by single stimulations. Venn diagrams display H2 Receptor custom synthesis responsive genes obtained following single therapy with LPS (A), BG (B) or 1,25 (OH)2D3 (125D) (C) in all models. Gene numbers in brackets represent the total quantity of genes discovered responsive to the indicated remedy, whilst gene numbers in bold highlight popular genes of all remedy circumstances. Genome-wide distribution of overlapping genes is monitored by Manhattan plots of log2FC values from 48 h remedies, that are obtained from model 1 for LPS (D) and BG (E) and model 2 for 1,25(OH)2D3 (F). Extremely CK2 Source prominent (absolute log2FC five) responsive genes are named and marked by colored dots, whereas the other genes are indicated by grey dots. Top rated five KEGG pathways representing essentially the most significantly enriched functions from the overlapping genes sorted by adjusted P-value (G ). Blue: LPS, purple: BG, red:1,25D. M1, model 1; M2, model two; M3, model three.”Toxoplasmosis”, “Cytokine-cytokine receptor interaction”, “Osteoclast differentiation” and “Fluid shear stress and atherosclerosis” had been found to become model-specific (Figures S2G ). Representative responsive genes were selected around the criteria i) becoming responsive to all remedies in a minimum of a single model ii)displaying prominent adjustments in expression and iii) getting involved inside the major KEGG pathways. The genes TMEM176A (transmembrane protein 176A), WNT5A (WNT family member 5A), CXCL1, S100A8 (S100 calcium binding protein A8), TNFSF15 (TNF superfamily member 15), CSF1 (colonyFrontiers in Immunology | frontiersin.orgDecember 2021 | Volume 12 | ArticleMalmberg et al.Vitamin D Remedy Sequence Is Criticalstimulating factor 1), CD163, INHBA, CCL1, MMP9, CDKN1A (cyclin dependent kinase inhibitor 1A) and TREM1 (triggering receptor expressed on myeloid cells 1) all represent previously reported LPS, BG or 1,25(OH)2D3 responsive genes (7, 40, 41) (Figure S5). They represent a 4×3 matrix indicating that the whole group of responsive genes could be classified into 12 categories, which include getting primarily responsive only to LPS or BG, each LPS and BG, or only 1,25(OH)2D3, as well getting all down- or upregulated or showing a mixed response. This highlighted intriguing specificities, for instance that CCL1 is clearly responsive both immune challenges however it barely responded to treatment with 1,25(OH)2D3, whereas TREM1 showed distinct preference for 1,25(OH) 2D3 . The examples with the mixed regulation category indicated that immune challenges led to improved gene expression when 1,25(OH)2D3 showed opposite regulation. In addition, model-specific variations had been observed, where, e.g., TNFSF15 showed distinct responsiveness although CSF1 responded almost the identical in all models. Taken together,