Repetitive Intermittent Hyperglycemia Drives the M1 Polarization and Inflammatory Responses in THP-1 Macrophages Through the Mechanism Involving the TLR4-IRF5 Pathway

Abstract

Repetitive intermittent hyperglycemia (RIH) is an independent risk factor for complications associated with type-2 diabetes (T2D). Glucose fluctuations commonly occur in T2D patients with poor glycemic control or following intensive therapy. Reducing blood glucose as well as glucose fluctuations is critical to the control of T2D and its macro-/microvascular complications. The interferon regulatory factor (IRF)-5 located downstream of the nutrient sensor toll-like receptor (TLR)-4, is emerging as a key metabolic regulator. It remains unclear how glucose fluctuations may alter the IRF5/TLR4 expression and inflammatory responses in monocytes/macrophages. To investigate this, first, we determined IRF5 gene expression by real-time qRT-PCR in the white adipose tissue samples from 39 T2D and 48 nondiabetic individuals. Next, we cultured THP-1 macrophages in hypo- and hyperglycemic conditions and compared, at the protein and transcription levels, the expressions of IRF5, TLR4, and M1/M2 polarization profile and inflammatory markers against control (normoglycemia). Protein expression was assessed using flow cytometry, ELISA, Western blotting, and/or confocal microscopy. IRF5 silencing was achieved by small interfering RNA (siRNA) transfection. The data show that adipose IRF5 gene expression was higher in T2D than nondiabetic counterparts (p = 0.006), which correlated with glycated hemoglobin (HbA1c) (r = 0.47/p < 0.001), homeostatic model assessment of insulin resistance (HOMA-IR) (r = 0.23/p = 0.03), tumor necrosis factor (TNF)-α (r = 0.56/p < 0.0001), interleukin (IL)-1β (r = 0.40/p = 0.0009), and C-C motif chemokine receptor (CCR)-2 (r = 0.49/p < 0.001) expression. IRF5 expression in macrophages was induced/upregulated (p < 0.05) by hypoglycemia (3 mM/L), persistent hyperglycemia (15 mM/L–25 mM/L), and RIH/glucose fluctuations (3–15 mM/L) as compared to normoglycemia (5 mM/L). RIH/glucose fluctuations also induced M1 polarization and an inflammatory profile (CD11c, IL-1β, TNF-α, IL-6, and monocyte chemoattractant protein (MCP)-1) in macrophages. RIH/glucose fluctuations also drove the expression of matrix metalloproteinase (MMP)-9 (p < 0.001), which is a known marker for cardiovascular complication in T2D patients. Notably, all these changes were counteracted by IRF5 silencing in macrophages. In conclusion, RIH/glucose fluctuations promote the M1 polarization and inflammatory responses in macrophages via the mechanism involving TLR4-IRF5 pathway, which may have significance for metabolic inflammation.

Ceramide kinase regulates TNF-α-induced immune responses in human monocytic cells

“…FACS data analysis was performed using BD FACSDiva Software 8 (BD Biosciences, San Jose, USA). The data for all flow cytometric measurements were expressed as staining index (SI) 20 which is the ratio of the separation between the positive population (vehicle in blue or treatment in pink) and the negative population (non-stained cells in grey), divided by two times the standard deviation of the negative population (non-stained cells in grey). The mean staining index was calculated from the three biological replicates minimum for each staining.…”

“…The blots were then washed four times with TBS and incubated for 2 h with HRP-conjugated secondary antibody (Promega, Madison, WI, USA). Immunoreactive bands were developed using an Amersham ECL plus Western Blotting Detection System (GE Health Care, Buckinghamshire, UK) and visualized by Molecular Imager ChemiDoc Imaging Systems (Bio-Rad Laboratories, Hercules, CA, USA) 22 – 24 .…”

Obesity, Nutrients and the Immune System in the Era of COVID-19

“…Thus, intermittent hyperglycemia is experimentally associated with an activation of myeloid lineages in the bone marrow, an increase in circulating pro-inflammatory monocytes and neutrophils [ 30 , 31 ], an increase in plasma concentrations of pro-inflammatory cytokines associated with increased oxidative stress, and endothelial dysfunction [ 32 ]. In vitro, intermittent exposure of macrophages to high glucose concentrations is responsible for a M1 polarization of these cells [ 33 ] by mechanisms associated with the activation of TLR4 and c-Jun N-terminal kinase (JNK) [ 34 ].…”

Involvement of Interleukin-1 Receptor-Associated Kinase 4 and Interferon Regulatory Factor 5 in the Immunopathogenesis of SARS-CoV-2 Infection: Implications for the Treatment of COVID-19

“…Crucially, therefore, blocking IRAK4 with a specific kinase inhibitor abolishes IRF5 activation but still permits NF-κB activation by other means, either by IKKβ itself via this or other signaling pathways (31), or using other kinases such as a MEKK3-dependent pathway (17,30,35,36). Speculatively, endosomal TLR3, responsive to dsRNA, may signal independently of MYD88 to IRF5 through TRAF6 using the adaptor TRIF (as well as to IRF3/7 via TRAF3) and may synergise with other TLRs (37,38,183); TLR4, translocated to endosomes, may also signal using TRAM-TRIF instead of MYD88 (19,(39)(40)(41)183). IRF5 homodimers complex with CBP/p300 to initiate the IRF5 transcriptome synergistically with NF-κB (42,43).…”

“…However, IRF5 binds strongly to the regulatory loci of other IRF5-targetted genes, such as IFNB, CXCL10, IL-10 (52), IL-12, and IL-23 (53), although in the case of anti-inflammatory IL-10, IRF5 is not directly responsible for its elevation in "cytokine storm, " being inhibitory at the IL-10 promoter (53,54). Mechanistically, a challenging complexity of variables influence IRAK4-IRF5 pathway activation outcomes (55): these include different IRF5 dimerization partners-including homodimerization and IRF7 (56); functionally different IRF5 isoforms, as investigated in plasmacytoid DCs (pDCs) (57); IRF5 interacting with different transcription factors (17), most critically the NF-κB subunits, p50 (48,58), and/or p65(RELA) (41,59,60); different cellular localizations, notably monocytes, macrophages, pDCs, and B cells (55, 58, 61); different triggers of pathway activation, for e.g., viral infection or autoimmunity; inhibition of the IRF5mediated activation of IFN-β by the IKKα pathway (62); and differences between murine and human cells (63)-all beyond the scope of this review. Nevertheless, despite these many complicating factors, the IRAK4-IRF5 axis consistently polarizes monocytes/macrophages toward the proinflammatory M1 (49,53,64) phenotype, displaying a similar innate cytokine/chemokine profile as in "cyokine storm" and indicating a potential therapeutic role for IRAK4 or IRF5 inhibition.…”