JUNB is a key transcriptional modulator of macrophage activation

Abstract

Activated macrophages are crucial for restriction of microbial infection but may also promote inflammatory pathology in a wide range of both infectious and sterile conditions. The pathways that regulate macrophage activation are therefore of great interest. Recent studies in silico have putatively identified key transcription factors that may control macrophage activation, but experimental validation is lacking. In this study, we generated a macrophage regulatory network from publicly available microarray data, employing steps to enrich for physiologically relevant interactions. Our analysis predicted a novel relationship between the AP-1 family transcription factor Junb and the gene Il1b, encoding the pyrogen IL-1β, which macrophages express upon activation by inflammatory stimuli. Previously, Junb has been characterized primarily as a negative regulator of the cell cycle, whereas AP-1 activity in myeloid inflammatory responses has largely been attributed to c-Jun. We confirmed experimentally that Junb is required for full expression of Il1b, and of additional genes involved in classical inflammation, in macrophages treated with LPS and other immunostimulatory molecules. Furthermore, Junb modulates expression of canonical markers of alternative activation in macrophages treated with IL-4. Our results demonstrate that JUNB is a significant modulator of both classical and alternative macrophage activation. Further, this finding provides experimental validation for our network modeling approach, which will facilitate the future use of gene expression data from open databases to reveal novel, physiologically relevant regulatory relationships.

Figure 1. Network analysis predicts a regulatory interaction between Junb and Il1b in macrophages

(A) Gene-gene relationships (edges) identified in the initial expression analysis (ARACNe) were enriched for biological relevance based on known interactions; the graph depicts the percent of edges remaining after each of two sequential refinements (triangular subnets and knowledge-enriched) that also appear in two databases of known biological interactions (InnateDB and HTRI). Numbers above bars indicate fold enrichment from the initial to the final datasets. (B) Regulatory network analysis identified significant covariance between Il1b and 7 transcription factors, including Rel and Junb. (C) Splenocytes from 4 week old mice (n = 4 of each genotype) were analyzed by flow cytometry to quantify frequencies of the indicated myeloid cells. RPMs, red pulp macrophages. cDC, conventional DC. pDC, plasmacytoid DC. (D-F) Macrophages were differentiated from the bone marrow of Junb^fl/fl or Junb^fl/fl x Lyz2 ^Cre/Cre (Junb^ΔLyz2) mice. (D) Deletion of Junb was assessed by quantitative PCR (qPCR) on BMDM genomic DNA. Means + SD for three technical replicates are shown. (E) BMDM from the indicated genotypes were stimulated with LPS for 4 h. Expression of JUNB (top panel) and β-actin (bottom panel) was measured in lysates by Western blot. (F) BMDM were labeled with antibodies to the indicated myeloid surface markers and analyzed by flow cytometry. Data shown are means + SD averaged from three independent experiments. (G) BMDM were incubated for 24 h with fluorescent beads of the indicated diameter, and phagocytosis was measured by flow cytometry. **, p < 0.01 by t-test.

Figure 2. JUNB regulates IL-1β production in macrophages treated with LPS

(A) Following 4 h treatment with LPS, Il1b mRNA levels were assessed by reverse transcription (RT) and qPCR. (B) BMDM were pre-treated for 3 h with LPS followed by addition of 5 mM ATP. IL-1β was measured in supernatant by ELISA. (C, D) BMDM were treated for 4 h with LPS, and intracellular pro-IL-1β was detected by flow cytometry. (C) Isotype staining and mock-treated controls were used to establish gates for F4/80⁺ BMDM. (D) The graph depicts the percent of BMDM positive for intracellular pro-IL-1β. (E) Peritoneal macrophages were stimulated for 2 h with LPS and stained for intracellular pro-IL-1β as in C, D. Data shown are means + SE (D, E) or SD (A, B) and are representative of three (A-D) or two (E) independent experiments. *, p < 0.05. **, p < 0.01. ***, p < 0.001 by t-test.

Figure 3. JUNB modulates expression of a cluster of immune-related genes in LPS-treated macrophages

(A-C) Junb^fl/fl or Junb^ΔLyz2 BMDM were treated for 4 h with LPS. RNA was harvested, amplified, and analyzed by microarray (A, B) or reverse transcribed into cDNA (C). (A) Genes exhibiting < 2-fold difference (black dots) or > 2-fold difference (red dots) in Junb^fl/fl versus Junb^ΔLyz2 macrophages. Results shown are averages from four technical replicates. (B) Histogram of fold-reduction in gene expression for JUNB-dependent genes. (C) Levels of the indicated transcripts were measured by RT-qPCR. Means + SD are shown. Representative results from one of three experiments are shown. **, p < 0.01. ***, p < 0.001 by t-tests. (D) Gating strategy for measurement of intracellular TNF. Isotype controls (not shown) were used as described for pro-IL-1β. (E) Induction of intracellular TNF after 4 h of LPS treatment. Bars represent means + SE for % of BMDM positive for TNF and are representative of three independent experiments.

Figure 4. JUNB affects late transcription of primary and secondary genes. (A)

Expression of AP-1 family members was assessed by microarray in BMDM with or without LPS stimulation as described in Fig. 3A. SD from 4 technical replicates is shown. (B) A subset of JUNB-dependent and JUNB-independent genes was identified by microarray and further classified into primary, secondary, tolerizable and non-tolerizable categories. +, Junb^fl/fl BMDM. ^Δ, Junb^ΔLyz2 BMDM. (C) Junb^fl/fl or Junb^ΔLyz2 BMDM were treated with LPS, and RNA was harvested at the indicated timepoints. Transcript of Il1b (left panel) or Il12b (right panel) was measured by RT-qPCR. Graphs depict means + SD and are representative of two independent experiments, each with three technical replicates.

Figure 5. JUNB regulates cytokine expression downstream of multiple pattern recognition receptors

(A-D) Junb^fl/fl or Junb^ΔLyz2 BMDM were treated for 4 h with the indicated ligands. Transcripts of the indicated cytokines were measured by RT-qPCR (A, B), and secreted protein was measured in the supernatant by cytometric bead array (C). (D) Production of pro-IL-1β and TNF by intracellular cytokine staining. Data are depicted as means + SD and are representative of two (C, D) or at least three (A, B) experiments. *, p < 0.05. **, p < 0.01. ***, p < 0.001 by t-tests.

Figure 6. JUNB modulates alternative activation markers in macrophages treated with IL- 4

(A) BMDM were treated with LPS for 4 h and gene expression was measured on microarrays, as described in Figure 3A. Statistical significance was as described in the methods. (B-E) BMDM were treated for 4 h (B, D, E) or 18 h (C) with IL-4. Levels of the indicated mRNA targets were measured by RT-qPCR (B, D, E) and arginase enzyme activity in cell lysates was assessed (C). Results shown are means + SD and are representative of three (B) or two (C, D, E) experiments. *, p < 0.05. **, p < 0.01. ***, p < 0.001 by t-tests.