Abstract
Tunneling nanotubes (TNTs) are cellular extensions enabling cytosol-to-cytosol intercellular interaction between numerous cell types including macrophages. Previous studies of hematopoietic stem and progenitor cell (HSPC) transplantation for the lysosomal storage disorder cystinosis have shown that HSPC-derived macrophages form TNTs to deliver cystinosin-bearing lysosomes to cystinotic cells, leading to tissue preservation. Here, we explored if macrophage polarization to either proinflammatory M1-like M(LPS/IFNγ) or anti-inflammatory M2-like M(IL-4/IL-10) affected TNT-like protrusion formation, intercellular transport and, ultimately, the efficacy of cystinosis prevention. We designed new automated image processing algorithms used to demonstrate that LPS/IFNγ polarization decreased bone marrow-derived macrophages (BMDMs) formation of protrusions, some of which displayed characteristics of TNTs, including cytoskeletal structure, 3D morphology and size. In contrast, co-culture of macrophages with cystinotic fibroblasts yielded more frequent and larger protrusions, as well as increased lysosomal and mitochondrial intercellular trafficking to the diseased fibroblasts. Unexpectedly, we observed normal protrusion formation and therapeutic efficacy following disruption of anti-inflammatory IL-4/IL-10 polarization in vivo by transplantation of HSPCs isolated from the Rac2−/− mouse model. Altogether, we developed unbiased image quantification systems that probe mechanistic aspects of TNT formation and function in vitro, while HSPC transplantation into cystinotic mice provides a complex in vivo disease model. While the differences between polarization cell culture and mouse models exemplify the oversimplicity of in vitro cytokine treatment, they simultaneously demonstrate the utility of our co-culture model which recapitulates the in vivo phenomenon of diseased cystinotic cells stimulating thicker TNT formation and intercellular trafficking from macrophages. Ultimately, we can use both approaches to expand the utility of TNT-like protrusions as a delivery system for regenerative medicine.
Highlights
Intercellular communication is essential to maintain and potentially restore homeostasis in multicellular organisms
We previously reported that co-culture of IC-21 macrophages with murine cystinotic fibroblasts appeared to increase the formation of TNTs7
We quantified the efficiency of lysosomal and mitochondrial transfer from the macrophages to the fibroblasts using another image analysis algorithm similar to the one we developed for Tunneling nanotubes (TNTs) quantification, which measures the frequency of Enhanced green fluorescent protein (eGFP) puncta (“donor”) within a region of interest (ROI) defined by DsRed+ “recipient” cell (Supplementary Fig. S6)
Summary
Intercellular communication is essential to maintain and potentially restore homeostasis in multicellular organisms. Due to cystinosin being a non-secreted transmembrane protein, the ubiquitous prevention of pathogenic cell damage using HSPCs was unexpected until the mechanism of trafficking was shown to involve TNT-mediated delivery of healthy lysosomes from HSPC-derived macrophages to diseased tissue[7]. Studies of HSPC transplantation as treatment for the mitochondrial neurodegenerative disorder Friedreich’s Ataxia reveal microglial correction of neurons, potentially through TNTs21 Taken together, these data indicate a prolific ability of phagocytic cells to widely disseminate therapeutic molecules via TNT trafficking. The plasticity of macrophages allows them to fulfill numerous biological functions ranging from proinflammatory roles in both arms of the immune system to immunomodulatory activities vis-à-vis tissue repair, homeostasis and resolution of inflammation These distinct phenotypic behaviors have been classified as either proinflammatory mediators of a type I immune response (M1 or classical activation) versus. This model risks oversimplification of a seamless phenotypic spectrum into a false dichotomy masking in vivo macrophage complexity, where these subtypes are dynamically changing in response to the cues received from the microenvironment, and never cleanly delineated[24,25]
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