Fig 1: HLJ1 deletion leads to the accumulation of homodimeric IL-12p35 and reduced levels of heterodimeric IL-12p70.(A) Bone marrow-derived macrophages (BMDMs) isolated from n = 6–7 Dnajb4+/+ and Dnajb4−/− mice were treated with 10 ng/ml lipopolysaccharide (LPS) and 20 ng/ml IFN-γ. Supernatant was collected at the indicated time points, and IL-12p70 was quantified via ELISA. 12 hr, p = 0.003; 24 hr, p = 0.003. (B) LPS/IFN-γ-treated BMDMs from n = 4–5 mice were lysed at the indicated time points and intracellular IL-12p70 was quantified via ELISA. 5 hr, p = 0.006; 8 hr, p = 0.012. (C) IL-12a, IL-12b, IL-6, and IL-18 expression was determined via quantitative real-time PCR (qRT-PCR) in LPS/IFN-γ-treated BMDMs isolated from n = 5 mice. (D) Intracellular IL-12p40 and HLJ1 expression levels were analyzed in LPS/IFN-γ-treated BMDMs isolated from Dnajb4+/+ (+/+) and Dnajb4−/− (−/−) mice. Representative samples of n = 3–5 biological replicates are shown. GAPDH served as a loading control. In comparisons with the 0 hr group (right panel): 2 hr, p = 0.001; 4 hr, p < 0.001; 8 hr, p = <0.001; 16 hr, p = 0.02. (E) The influence of human HLJ1 knockdown on the redox state of human IL-12p35 was analyzed via non-reducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). 293T cells were (co-)transfected with the indicated IL-12p35 subunits and shRNA targeting HLJ1. The percentage of high-molecular-weight (HMW) and low-molecular-weight (LMW) IL-12p35 species in the presence or absence of shHLJ1 was quantified (right panel, n = 4 biological repeats for shHLJ1- and control-transfected cultures; p = 0.001). Where indicated, samples were treated with β-mercaptoethanol (β-Me) after cell lysis to provide a standard for completely reduced protein. GAPDH served as a loading control. Data presented are means ± standard deviation (SD). Statistical analysis was performed by using the two-tailed, unpaired Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001. Figure 9—source data 1.Data for graphs depicted in Figure 9A–E. Figure 9—source data 2.Original and labeled blots images of Figure 9D, E.
Fig 2: C1q deletion is associated with increased levels of pro-inflammatory cytokines in the PFC.IL-23. Two-way ANOVA, genotype X treatment interaction (F (1, 23) = 0.06640, p = 0.7989, n = 6–7); Tukey’s multiple comparisons; $$p = 0.0056 vs no-shock WT, ##p = 0.0094 vs shock WT. IL-1α. Two-way ANOVA, genotype X treatment interaction (F (1, 23) = 0.2762, p = 0.6043, n = 6–7); Tukey’s multiple comparisons- no significant difference between the groups. IFN-γ. Two-way ANOVA, genotype X treatment interaction (F (1, 23) = 2.744, p = 0.1112, n = 6–7); Tukey’s multiple comparisons; $$$$p < 0.0001 vs no-shock WT, ##p = 0.0081 shock WT. TNF-α. Two-way ANOVA, genotype X treatment interaction (F (1, 23) = 0.2117, p = 0.6497, n = 6–7); Tukey’s multiple comparisons; $$$p = 0.0002 vs no-shock WT, ###p = 0.0005 shock WT. MCP-1. Two-way ANOVA, genotype X treatment interaction (F (1, 20) = 0.4140, p = 0.5273, n = 5–7); Tukey’s multiple comparisons; $$$p = 0.0010 vs no-shock WT, #p = 0.0165 vs shock WT. IL-12p70. Two-way ANOVA, genotype X treatment interaction (F (1, 23) = 1.207, p = 0.2833, n = 6–7); Tukey’s multiple comparisons; $$p = 0.0013 vs no-shock WT, #p = 0.0342 vs shock WT. IL-1β. Two-way ANOVA, genotype X treatment interaction (F (1, 23) = 0.5080, p = 0.4832, n = 6–7); Tukey’s multiple comparisons—no significant difference between the groups. IL-17α. Two-way ANOVA, genotype X treatment interaction (F (1, 23) = 0.0573, p = 0.8128, n = 6–7); Tukey’s multiple comparisons; $$p = 0.0053 vs no-shock WT, ##p = 0.0016 vs shock WT. GM-CSF. Two-way ANOVA, genotype X treatment interaction (F (1, 23) = 1.087, p = 0.3079, n = 6–7); Tukey’s multiple comparisons; $$$p = 0.0009 vs no-shock WT, #p = 0.0206 vs shock WT. IL-27. Two-way ANOVA, genotype X treatment interaction (F (1, 24) = 0.5738, p = 0.4561, n = 7); Tukey’s multiple comparisons; $$$$p < 0.0001 vs no-shock WT, ####p < 0.0001 vs shock WT. INF-β. Two-way ANOVA, genotype X treatment interaction (F (1, 24) = 1.407, p = 0.2472, n = 6–7); Tukey’s multiple comparisons; $p = 0.0114 vs no-shock WT. Data are shown as mean ± SEM.
Fig 3: Lower dietary protein intake into middle age results in kidney leukocyte infiltration and inflammation(A) Representative photomicrographs of leukocyte infiltration in periodic acid-Schiff (PAS)-stained kidney cortices according to score 1–3 (×200 magnification) (arrows indicate regions of leukocyte infiltrate).(B) Ordinal regression was used to model the relationship between leukocyte infiltration and macronutrient intake, and plots show the probability of obtaining a score of 1 (red, low infiltration), 2 (green, moderate infiltration), or 3 (blue, high infiltration) as protein (left panel), CHO (middle panel), and fat (right panel) intake increased (n = 160).(C) Kidney leukocyte infiltration scores presented by tertiles of dietary protein intake (low < 6.5 kJ/mouse/day, med 6.5–12 kJ/mouse/day, high 12.1–30 kJ/mouse/day).(D) Kidney leukocyte infiltration scores presented by tertiles of dietary fat intake (low <8.58 kJ/mouse/day, med 8.59–13.8 kJ/mouse/day, high 13.8–44.9 kJ/mouse/day).(E) Representative photomicrographs of regions of the renal cortex with leukocyte infiltrate (×800) stained for T cell markers CD3, CD4, FoxP3.(F) Summarized p values for coefficients for the GAMs of kidney cytokine profiling using Legendplex 13-plex pro-inflammatory cytokine array. p values for individual effects (single column) or interactive effects (spanning 2 or more columns) of macronutrients on cortical cytokine profiles are shown (NS, non-significant; n numbers and full p values in Table S1).(G–J) Response surfaces showing the effect of macronutrient intake on kidney cortex concentrations of select inflammatory cytokines (pg/μg of total protein). (G) IL-23, (H) IL-1β, (I) IFNγ, (J) IL-12p70.
Fig 4: Gene expression heatmaps show altered innate and adaptive biomarker expression in 4T1 tumors from Control and INDO-treated WT and Nos2- mice. A) 4T1 tumors from WT and Nos2-mice treated with INDO showed increased DC, T cell, Ifn, and macrophage-associated gene signatures. Tumors from Nos2-mice showed increased N1 neutrophils, B cell activation biomarkers, and Ig-associated gene signatures. B) Gene expression quantification showing significantly increased CD8a, Ifnγ, Tbx21, Gzmb, and reduced IL10 in INDO-treated mice, which implicate increased cytolytic CD8+ T cell function. Heatmaps and their color scale were generated per gene across different comparison pairs using the fold change values obtained from the differential gene detection algorithm, gene-specific analysis (GSA) in Partek Flow with default settings. Max and Min on the Counts color scale bar are used to indicate the direction of fold change. WT (n = 7, gray), INDO (n = 8, yellow), Nos2- (n = 7, red), INDO + Nos2- (n = 8, orange).*p ≤ 0.01, **p ≤ 0.001, ***p ≤ 0.0001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig 5: Absence of DC-STAMP decreased the expression of pro-inflammatory and antiinflammatory cytokines in macrophages from Tg(hTNF) mice. (A) mRNA expression for Nos2, Tnf, Il-16 and 1l-10 was estimated by RT-PCR in WT, Dcstamp-/-, Tg(hTNF) and Dcstamp-/-;Tg(hTNF) CD11b+ adherent macrophages stimulated with the M2 polarizing cytokine IL-4 or the M1 polarizing factors: IFNY + LPS. (B) Quantification of IL1B, TNF, IL6 and CCL2 in supernatants collected at 72 h from cultures of adherent macrophages. Graphs represent the mean ± SD (n = 3). ns, not statistically significant,*p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001 (Two-way ANOVA and paired Student's t-test).
Supplier Page from BioLegend for LEGENDplex™ MU Inflam Panel (13-plex) w/ VbP