Fig 2: A map for the putative binding sites of transcription factors on the promoter of TNFA and IL1B and relative binding activity of NF-?B on the promoter of TNFA and IL1B. a Numbers refer to the position relative to the transcriptional starting site, indicated by black arrows. The position of the transcription factors is indicated by the respective symbols. The positions of primers used for chromatin immunoprecipitation assays are denoted by dark lines surround the symbol of NF-?B. The identification for target promoter regions of candidate genes were determined by BLAST analyses as DNA-sequences that are 5'-upstream of the mRNA sequences deposited in the NCBI: NM_173966.3 (TNFA), NM_174093.1 (IL1B). b Level of NF-?B binding to the TNFA and IL1B promoter. Six individual samples in each group were involved in generating chromatin immunoprecipitation analysis. The letters in superscript indicate that the difference between groups was significant (P < 0.05)

Fig 3: NMI stimulates macrophages through the TLR4 pathway. a Western blot analysis of mNMI in the BMDM cell lysate after 1 h incubation with recombinant mNMI. The BMDM cells were isolated from Nmi -/- mice and pretreated with macrophage colony-stimulating factor (MCSF). b, c TNF and IL-6 released by BMDMs from WT (C57BL/6) mice, pretreated with bafilomycin A1 (10 nM), TAK-242 (100 nM) or dimethyl sulphoxide (DMSO) for 2 h and stimulated with mNMI (5 µg ml-1) or LPS (100 ng ml-1) for 8 h. d–g TNF and IL-6 levels in the supernatants of BMDMs from WT, Tlr4 -/- and Tlr2 -/- mice were analyzed using ELISA 4 h post activation by different stimulus. h After incubation with mNMI-GFP for 1 h, the percentage of GFP labeled CD11b+F4/80+ cells was determined by Flow cytometric analysis. The cells were isolated from spleen in WT or Tlr4 -/- mice. i NMI in the human THP1 cell (ATCC TIB-202™) lysates interacts with hTLR4. Ni-NTA beads coupled with 2 µg His-hTLR4 and/or His-hMD2 fusion proteins were used as bait. j The luciferase activity of HEK293T cells (ATCC CRL-11268™) are shown after stimulated with 5 µg ml-1 mNMI for 4 h (in the presence or absence of 25 µg ml-1 polymyxin B (PMB)). The cells were pre-transfected with mTLR4-MD2-CD14 and NF-?B promoter with luciferase activity. 100 ng ml-1 LPS was administrated as positive control. In b–e and g, error bars indicate ± s.e.m. from 3 biological replicates. Significance was tested by one-way ANOVA followed by Student–Newman–Keuls test. **P < 0.01

Fig 4: Acidosis effect on inflammation markers in NRK-49F cells in mono- and co-cultures. Impact of acidosis on TGF-ß (a,d), TNF (b,e), COX-2 (c,f). Representative Western blots of proteins isolated from cells exposed to acidic media (g). p < 0.05 at significant difference compared with the control group; n - (8 - 19). exposure time = 48 h.

Fig 5: CD1c+ CD14+ mononuclear cells contain Nef and secrete inflammatory cytokines.FACS and immunohistochemistry analysis of Nef-containing mononuclear cells. (a) Assessment of CD1c+ CD14+ cells in CD1c+ positive selected PBMC from 2 non-viremic patients and a healthy control. Aliquots of the cells were stained for Nef and CD14 by immunohistochemistry. (b) Percentage of CD1c+ CD14+ cells in the CD1c+ CD19- cell fraction based on the analysis of 5 non-viremic patients and healthy controls. Error bars represent mean ± SEM. (c) Secretion of TNF, IL-6 and IFN? by 105 PBMC obtained from 4 non-viremic patients and controls in 12 h. PBMC were left untreated or the CD1c+ fraction was positive depleted (by magnetic beads) (d) TNF and IL-6 secretion from PBMC, or from 105 positive-selected CD14+ and CD19+ cells in 12 h. Error bars show the SEM.