Abstract
Macrophages possess mechanisms for reinforcing the integrity of their endolysosomal membranes against damage. This property, termed inducible renitence, was previously reported for macrophages stimulated with LPS, peptidoglycan, IFN-γ, or TNF-α. Here, we expanded the macrophage subtypes examined to include populations with well-defined functional roles in vivo: classically activated macrophages (CA-Mφ), alternatively activated macrophages (AA-Mφ), and regulatory macrophages (Reg-Mφ). We determined that renitence is a property of CA-Mφ and Reg-Mφ, but not of AA-Mφ. Furthermore, LPS-activated macrophages possess features of both CA-Mφ and Reg-Mφ, based on their cytokine secretion profiles. As the generation of these three classes of renitent macrophages required exposure to LPS, a Toll-like receptor (TLR) ligand, we assessed whether TLR stimulation generally induced renitence. Stimulation of TLRs 2/1, 3, and 4 induced renitence, whereas stimulation of TLRs 7/8 and 9 induced modest levels of lysosomal damage protection. Renitence induced by TLR stimulation required the signaling adaptors MyD88 and TRIF. Surprisingly, the specific signaling adaptor usage requirements for some TLRs differed from those established for canonical TLR signaling. Of note, renitence induced by LPS, a TLR4 ligand, required signaling through TRIF but not MyD88. Consistent with this pattern, the type I IFN response, which is triggered by LPS stimulation through a TRIF-dependent, MyD88-independent pathway, contributed to renitence. A biologically relevant type I IFN trigger in macrophages, murine norovirus-1 (MNV-1) infection, also induced renitence. This work establishes the concept that susceptibility to lysosomal damage within macrophages varies according to inflammatory state and depends on the type I IFN response.
Summary sentence Macrophages of distinct polarization states exhibit varying degrees of susceptibility to lysosomal damage reflecting their functional roles in host defense and immune regulation
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
Abbreviations
A2bR, adenosine 2b receptor; AA-Mφ, alternatively activated macrophages; Ado, adenosine; ANOVA, analysis of variance; ATP, adenosine triphosphate; AW, acid-washed; BMM, bone marrow-derived macrophages; CA-Mφ, classically activated macrophages; dsRNA, doublestranded RNA; ELISA, enzyme-linked immunosorbent assay; FBS, fetal bovine serum; Fdx, fluorescein dextran; FLA-ST, flagellin from Salmonella typhimurium; Gapdh, glyceraldehyde 3-phosphate dehydrogenase; IFITM3, interferon-induced transmembrane protein 3; IFN, interferon; IFN-γ, interferon-γ; Ifnar1, interferon-α/β receptor 1; IL-4, interleukin-4; IL-10, interleukin-10; IL-13, interleukin-13; L. monocytogenes, Listeria monocytogenes; ISG, interferon-stimulated gene; LPS, lipopolysaccharide; M-CSF, macrophage colony-stimulating factor; MDA5, melanoma differentiation-associated protein 5; MNV-1, murine norovirus-1; MOI, multiplicity of infection; MyD88, myeloid differentiation primary response 88; NCOA, nuclear receptor coactivator 7; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; Pam3CSK4, Pam3CysSerLys4; PGE2, prostaglandin E2; poly(I:C), polyinosinic:polycytidylic acid; qPCR, quantitative polymerase chain reaction; R848, Resiquimod; Reg-Mφ, regulatory macrophages; RelA, nuclear factor NF-kappa-B p65 subunit; Relm-α, resistin-like molecule-α; RIG-I, retinoic acid-inducible gene I; ssRNA, single-stranded RNA; TLR, Toll-like receptor; TNF-α, tumor necrosis factor-α; TRIF, TIR (Toll-interleukin-1 receptor) domain-containing adaptor-inducing interferon-β; WT, wild-type.
