CD38 Is Robustly Induced in Human Macrophages and Monocytes in Inflammatory Conditions

Abstract

Macrophages and their monocyte precursors mediate innate immune responses and can promote a spectrum of phenotypes from pro-inflammatory to pro-resolving. Currently, there are few markers that allow for robust dissection of macrophage phenotype. We recently identified CD38 as a marker of inflammatory macrophages in murine in vitro and in vivo models. However, it is unknown whether CD38 plays a similar marker and/or functional role in human macrophages and inflammatory diseases. Here, we establish that CD38 transcript and protein are robustly induced in human macrophages exposed to LPS (±IFN-γ) inflammatory stimuli, but not with the alternative stimulus, IL-4. Pharmacologic and/or genetic CD38 loss-of-function significantly reduced the secretion of inflammatory cytokines IL-6 and IL-12p40 and glycolytic activity in human primary macrophages. Finally, monocyte analyses in systemic lupus erythematosus patients revealed that, while all monocytes express CD38, high CD38 expression in the non-classical monocyte subpopulation is associated with disease. These data are consistent with an inflammatory marker role for CD38 in human macrophages and monocytes.

Keywords: CD38; macrophage; marker; monocyte; systemic lupus erythematosus.

Figure 1

CD38 mRNA and protein expression is increased in human monocytic cell lines differentiated into M(LPS + IFN-γ) macrophages. Expression of CD38 and FPR2 mRNA in THP-1 (A,B) and U937-derived macrophages (C,D) in unstimulated (M0), M(LPS + IFN-γ)-stimulated (labeled M1 throughout figure), or M(IL-4)-stimulated (labeled M2 throughout figure) macrophages was measured by real-time PCR and expressed as mean relative expression ± SD (n = 3 biological replicates with two technical replicates per sample). Gene expression is expressed as fold change ± SD relative to M0 condition. One-way analysis of variance with p values adjusted for multiple comparisons using Sidak’s multiple comparisons test compare M(LPS + IFN-γ) vs. M0 and M(LPS + IFN-γ) vs. M(IL-4). (E,F) Flow cytometry staining of surface FPR2 on x-axis, CD38 on y-axis in THP-1 (E) and U937 (F) cells. Flow plots correspond to total cells. Data shown are representative of n = 3 biological replicates. (G–J) Quantification of CD38⁺ cells and FPR2⁺ cells in THP-1 (G,H) or U937 cells (I,J) are expressed as percent of positive cells ± SD (n = 3 biological replicates); ISO, isotype control; **p < 0.01, ****p < 0.0001.

Figure 2

Increased CD38 expression in human M(LPS + IFN-γ) monocyte-derived macrophages (MDMs). Expression of human CD38 (A), FPR2 (B), GPR18 (C), EGR2 (D), and c-MYC (E) genes in human MDMs in unstimulated (M0), M(LPS + IFN-γ) (labeled M1 throughout figure), or M(IL-4) (labeled M2 throughout figure) human MDMs, as measured by real-time PCR. Gene expression level is expressed as fold change ± SD relative to M0 condition (n = 7–8 independent samples, each generated from different donors; each independent sample value corresponds to the average of two technical replicates). One-way analysis of variance (ANOVA) with p values adjusted for multiple comparisons using Sidak’s multiple comparisons test, *p < 0.05, **p < 0.01, ****p < 0.0001. (F) Flow cytometry staining of surface CD38 and CD40 at 24 h post-differentiation into M0, M(LPS + IFN-γ), or M(IL-4) macrophages. Flow plots correspond to CD11b⁺ cells. Data shown are representative of n = 4 independent samples generated from different individual donors; ISO, isotype control. (G,H) Quantification of CD38⁺ cells (G) or CD40⁺ cells (H) is expressed as percent of cells ± SD (n = 4 independent samples generated from different individual donors). One-way ANOVA with p values adjusted for multiple comparisons using Sidak’s multiple comparisons test, **p < 0.01, ***p < 0.001, ****p < 0.0001. (I) Quantification of flow cytometry staining of surface CD38⁺ cells at 24 h post-differentiation into M0, M(LPS + IFN-γ), M(LPS), M(IFN-γ), M(IL-1β), M(TNF-α), M(TLR7), or M(TLR8) macrophages is expressed as percent of cells ± SD (n = 3 samples from MDM generated in three independent experiments from three distinct donors, with two technical replicates per sample). One-way ANOVA with p values adjusted for multiple comparisons using Dunnett’s multiple comparisons test, ****p < 0.0001. (J) Pearson’s correlation analysis between CD38⁺ cells and IL-1β⁺ cells. The straight line represents linear regression calculation, r = 0.5512, Pearson’s **p < 0.01.

Figure 3

CD38 promotes inflammatory cytokine secretion in human macrophages. Human monocyte-derived macrophages (MDMs) were treated with 15 µM rhein or DMSO control (A), 25 µM apigenin or DMSO (B), or transfected with 100 µM CD38 siRNA cocktail or siRNA control (C) on day 5. On day 6, they were activated with LPS + IFN-γ for an additional 24 h prior to analysis. (A–C) IL-6 and IL-12p40 secretion was analyzed by ELISA from MDM supernatants. Graphs pool normalized (relative to corresponding experiment vehicle control or nonsense siRNA) data from three (A), five (B), or six (C) independent experiments/donors, with at least two technical replicates for each sample. Data are expressed as mean cytokine secretion ± SD relative to vehicle condition. (D) Quantification of CD38⁺ cells analyzed by flow cytometry from MDM transfected with control or CD38 siRNAs for data shown in panel (C) is expressed as percent of cells ± SD, n = 6 independent experiments/donors and two technical replicates per sample. (E) Quantification of IL-1β⁺ MDMs analyzed by flow cytometry for data shown in panel (C) is expressed as percent of cells ± SD, n = 6 independent experiments/donors and two technical replicates per sample. (F) l-Lactate assays run using supernatants from panel (C), n = 5 independent experiments/donors, with at least three technical replicates per sample. All data were analyzed by unpaired t tests. **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 4

CD38 mean fluorescence intensity (MFI) in non-classical monocytes (NCMs) is elevated in active systemic lupus erythematosus (SLE). (A) Gating strategy: peripheral blood mononuclear cells isolated from healthy donors (HD) or SLE patients (panel 1) were gated for CD45⁺ cells (panel 2) prior to gating out CD66b⁻ cells (panel 3) and CD3⁻CD19⁻CD56⁻ (panel 4) cells. The remaining population was analyzed with CD14 and CD16 after removing cells that are neither CD14⁺ nor CD16⁺ (NOT gate) (panel 5) to identify classical (CD14⁺⁺CD16⁻) (CM), intermediate (CD14⁺CD16⁺) (IM) and non-classical (CD14⁺CD16⁺) (NCM) monocytes (panel 6). A stricter gate that excluded CD14⁻ cells was also used for some analyses (box in panel 6 labeled CD14^lowCD16⁺⁺). (B) Example of how the relative percentage of CM, IM, and NCM among monocytes was calculated and how CM, IM, and NCM populations were analyzed for CD38 expression. ISO, isotype. (C) Percent of CD38⁺ cells within the CM, IM and NCM subsets in HD, inactive (SLE^I = SLEDAI 0), or active (SLE^A = SLEDAI > 4) patients. (D) A histogram of CD38 expression in NCM indicating how the CD38^hi subset was defined. A histogram from each group is overlaid, including an SLE^I patient with an SLE Disease Activity Index (SLEDAI) of 0 and an SLE^A patient with an SLEDAI of 16. (E) CD38 MFI within CD38⁺ CM, IM, and NCM populations of HD, SLE^I, and SLE^A patients. (F) Percent of CD38^hi cells within CM, IM, and NCM subsets in HD, SLE^I, or SLE^A patients. (G) CD38 MFI within CD38⁺ CD14^lowCD16⁺⁺ population in HD, SLE^I, or SLE^A patients. (H) Percent of CD38^hi cells within CD14^lowCD16⁺⁺ population in HD, SLE^I, or SLE^A patients. (C,E–H) Quantification of CD38⁺ cells (C) and CD38^hi cells (F,H) is expressed as percent of positive cells ± SD. Quantification of CD38 MFI (E,G) is expressed as MFI of positive cells ± SD. n = 9 donors for HD, n = 11 patients for SLE^I, and n = 10 patients for SLE^A. Each sample was run in duplicate, and duplicate values were averaged prior to analysis. One-way analysis of variance with p values adjusted for multiple comparisons using Tukey’s multiple comparisons test, *p < 0.05, **p < 0.01. (I,J) Correlation analyses of SLEDAI score with NCM CD38 MFI (I) or percent of CD38^hi NCM (J) in active and inactive SLE patients. (I) CD38 MFI has a Gaussian distribution as determined by D’Agostino and Pearson normality test, so Pearson correlation analysis was performed, r = 0.6265, **p < 0.005. (J) Percent of CD38^hi cells does not have a Gaussian distribution as determined by D’Agostino and Pearson normality test, so Spearman correlation analysis was performed, r = 0.6085, **p < 0.005.