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. 2019 Feb 1;202(3):920-930.
doi: 10.4049/jimmunol.1800226. Epub 2018 Dec 28.

IRF5 Is Required for Bacterial Clearance in Human M1-Polarized Macrophages, and IRF5 Immune-Mediated Disease Risk Variants Modulate This Outcome

Matija Hedl  1 , Jie Yan  1 , Heiko Witt  2 , Clara Abraham  3
Affiliations

IRF5 Is Required for Bacterial Clearance in Human M1-Polarized Macrophages, and IRF5 Immune-Mediated Disease Risk Variants Modulate This Outcome

Matija Hedl et al. J Immunol. .

Abstract

Common IFN regulatory factor 5 (IRF5) variants associated with multiple immune-mediated diseases are a major determinant of interindividual variability in pattern recognition receptor (PRR)-induced cytokines in macrophages. PRR-initiated pathways also contribute to bacterial clearance, and dysregulation of bacterial clearance can contribute to immune-mediated diseases. However, the role of IRF5 in macrophage-mediated bacterial clearance is not well defined. Furthermore, it is unclear if macrophages from individuals who are carriers of low IRF5-expressing genetic variants associated with protection for immune-mediated diseases might be at a disadvantage in bacterial clearance. We found that IRF5 was required for optimal bacterial clearance in PRR-stimulated, M1-differentiated human macrophages. Mechanisms regulated by IRF5 included inducing reactive oxygen species through p40phox, p47phox and p67phox, NOS2, and autophagy through ATG5. Complementing these pathways in IRF5-deficient M1 macrophages restored bacterial clearance. Further, these antimicrobial pathways required the activation of IRF5-dependent MAPK, NF-κB, and Akt2 pathways. Importantly, relative to high IRF5-expressing rs2004640/rs2280714 TT/TT immune-mediated disease risk-carrier human macrophages, M1-differentiated GG/CC carrier macrophages demonstrated less reactive oxygen species, NOS2, and autophagy pathway induction and, consequently, reduced bacterial clearance. Increasing IRF5 expression to the rs2004640/rs2280714 TT/TT levels restored these antimicrobial pathways. We define mechanisms wherein common IRF5 genetic variants modulate bacterial clearance, thereby highlighting that immune-mediated disease risk IRF5 carriers might be relatively protected from microbial-associated diseases.

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Figures

Figure 1.
Figure 1.. IRF5 is required for optimal intracellular bacterial clearance.
(A) Timeline schematic for M1 polarization, IRF5 knockdown and LPS treatment. (B) MDMs (n=6 donors, similar results in n=12–16) were left untreated (M0) or polarized into M1 macrophages and then transfected with scrambled or IRF5 siRNA. Cells were then left untreated or treated with 0.1μg/ml LPS for 48h and co-cultured with E. faecalis, AIEC, S. aureus or S. Typhimurium. Colony forming units (CFU)+SEM. Significance is compared to scrambled siRNA-transfected cells for each respective condition (i.e. M0, M1 and M1+LPS) or as indicated. Scr, scrambled; **, p<0.01; ***, p<0.001; †, p<1×10−4; ††, p<1×10−5.
Figure 2.
Figure 2.. IRF5 is required for optimal LPS-induced MAPK, NFκB and Akt2 pathway activation in M1 macrophages, and these pathways are required for enhanced bacterial clearance in LPS-treated M1 macrophages.
(A-C) MDMs (n=6 donors, similar results seen in an independent n=6) were polarized into M1 macrophages, transfected with scrambled or IRF5 siRNA, and then treated with 0.1μg/ml LPS for 15 min. Summarized data are represented as the fold (A) phospho-ERK, phospho-p38, phospho-JNK, (B) phospho-IκBα, or (C) phospho-Akt2 change normalized to untreated cells (represented by the dotted line at 1) + SEM. (D) MDMs were treated as in (A-C) and the indicated proteins assessed by Western blot. (E) MDMs (n=6, similar results in n=12 for MAPK/NEMO and full combination) were polarized into M1 macrophages, transfected with scrambled or combined ERK, p38, JNK (MAPK), NEMO (NE), or Akt2 siRNA alone or in various combinations as indicated, then left untreated or treated with 0.1μg/ml LPS for 48h and co-cultured with E. faecalis, AIEC, S. aureus or S. Typhimurium. CFU+SEM. Significance is compared to scrambled siRNA-transfected, LPS-treated cells or as indicated. Scr, scrambled; p-, phospho-; tx, treatment. **, p<0.01; ***, p<0.001; †, p<1×10−4; ††, p<1×10−5.
Figure 3.
Figure 3.. IRF5 is required for optimal induction of NADPH oxidase subunits and NOS2 which contribute to bacterial clearance in PRR-stimulated M1 macrophages.
(A-D) MDMs were polarized into M1 macrophages, transfected with scrambled or IRF5 siRNA, and then left untreated or treated with 0.1μg/ml LPS for 48h. (A) Representative flow cytometry for the ROS-detecting dye H2DCFDA with MFI values and summary graph with MFI+SEM (n=6 donors). (B) Summary graph for nitroblue tetrazolium+SEM (n=6). (C) p40phox, p47phox, p67phox, gp91phox, p22phox and (D) NOS2 mRNA expression. Fold mRNA change compared to scrambled siRNA-transfected, untreated cells+SEM (n=6). (E-G) MDMs were polarized into M1 macrophages, transfected with scrambled or the indicated siRNA, and then treated with 0.1μg/ml LPS for 48h. p40phox, p47phox, p67phox, and NOS2 protein expression was assessed by: (E) Western blot or (F) flow cytometry with MFI+SEM (n=6). (G) Cells were cultured with E. faecalis. CFU+SEM (n=8). (H-K) MDMs were polarized into M1 macrophages, transfected with scrambled or IRF5 siRNA±p47phox-, p67phox- or NOS2-expressing vectors or empty vector (EV) then left untreated or treated with 0.1μg/ml LPS for 48h. (H) p47phox and p67phox protein (n=6), (I) ROS (n=6), and (J) NOS2 protein (n=6) expression was assessed by flow cytometry. MFI+SEM. (K) Cells (n=8, similar results in an independent n=4) were cultured with E. faecalis. CFU+SEM. Significance is compared to IRF5 siRNA, EV-transfected cells or as indicated. Tx, treatment; scr, scrambled. **, p<0.01; ***, p<0.001; †, p<1×10−4; ††, p<1×10−5.
Figure 4.
Figure 4.. IRF5 is required for optimal induction of the autophagy protein ATG5, thereby contributing to bacterial clearance in PRR-stimulated M1 macrophages.
(A-C) MDMs were polarized into M1 macrophages, transfected with scrambled or IRF5 siRNA, and then treated with 0.1μg/ml LPS for 48h. (A-B) LC3II protein expression was assessed by: (A) Western blot or (B) flow cytometry with representative plot and summary graph of MFI+SEM (n=6 donors). (C) ATG5, ATG7, and IRGM fold mRNA induction compared to scrambled siRNA-transfected, untreated cells+SEM (n=6). (D-H) MDMs were polarized into M1 macrophages, transfected with scrambled or ATG5 siRNA, then treated with 0.1μg/ml LPS for 48h. ATG5 and LC3II protein expression was assessed by: (D,F) Western blot, or (E,G) flow cytometry with MFI+SEM (n=6). (H) Cells were cultured with E. faecalis. CFU+SEM (n=8). (I-K) MDMs were polarized into M1 macrophages, transfected with scrambled or IRF5 siRNA± ATG5-expressing vector or empty vector (EV), then treated with 0.1μg/ml LPS for 48h. (I) ATG5 (n=6) and (J) LC3II (n=6) protein expression was assessed by flow cytometry with MFI+SEM. (K) Cells (n=8, similar results in an independent n=4) were cultured with E. faecalis. CFU+SEM. Scr, scrambled; tx, treatment. ***, p<0.001; †, p<1×10−4; ††, p<1×10−5.
Figure 5.
Figure 5.. ROS, RNS and autophagy pathways cooperate for optimal IRF5-dependent bacterial clearance.
(A) MDMs (n=4 donors) were transfected with scrambled or the indicated siRNA, polarized into M1 macrophages, then treated with 0.1μg/ml LPS for 48h. (B) MDMs (n=8) were polarized into M1 macrophages, transfected with scrambled or IRF5 siRNA±p47phox-, p67phox-, NOS2-, or ATG5-expressing vectors alone or in combination, or empty vector (EV), then treated with 0.1μg/ml LPS for 48h. (A-B) Cells were cultured with E. faecalis, AIEC, S. aureus and S. Typhimurium. CFU+SEM. Significance is compared in ‘A’ to scrambled siRNA-transfected cells, or in ‘B’ to IRF5 siRNA, EV-transfected cells or as indicated. Scr, scrambled. *, p<0.05; **, p<0.01; ***, p<0.001; †, p<1×10−4; ††, p<1×10−5.
Figure 6.
Figure 6.. MDMs and M1 macrophages from immune-mediated disease-protective rs2004640/rs2280714 GG/CC carriers show reduced intracellular bacterial clearance and ROS, RNS and autophagy pathway induction.
MDMs from rs2004640/rs2280714 TT/TT, GT/CT and GG/CC carriers (n=15/genotype) were left undifferentiated or were differentiated under M1 polarizing conditions and then treated with 0.1 μg/ml LPS for 48h. (A,F) IRF5 expression was examined by flow cytometry. MFI+SEM. (B,G) Cells were cultured with E. faecalis or S. Typhimurium. CFU+SEM. (C-E, H-J) Summarized MFI of ROS or the indicated proteins as assessed by flow cytometry+SEM. *, p<0.05; **, p<0.01; ***, p<0.001; †, p<1×10−4; ††, p<1×10−5.
Figure 7.
Figure 7.. Rs2004640/rs2280714 genotype-dependent regulation of bacterial clearance, ROS, RNS and autophagy proteins is due to IRF5 expression modulation.
MDMs from rs2004640/rs2280714 TT/TT or GG/CC carriers (n=15/genotype) were transfected with 25 nM scrambled or IRF5 siRNA (A-E), or empty vector or IRF5 vector (F-J), and then polarized to M1 macrophages and treated with 0.1μg/ml LPS for 48h. (A,F) Summarized MFI of IRF5 as assessed by flow cytometry+SEM. (B,G) Cells were cultured with E. faecalis or S. Typhimurium. CFU+SEM. (C-E, H-J) Summarized MFI of ROS or the indicated proteins as assessed by flow cytometry+SEM. NS, not significant; scr, scrambled. ††, p<1×10−5.
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