IRE1α promotes phagosomal calcium flux to enhance macrophage fungicidal activity

Abstract

The mammalian endoplasmic reticulum (ER) stress sensor inositol-requiring enzyme 1α (IRE1α) is essential for cellular homeostasis and plays key roles in infection responses, including innate immunity and microbicidal activity. While IRE1α functions through the IRE1α-XBP1S axis are known, its XBP1S-independent roles are less well understood, and its functions during fungal infection are still emerging. We demonstrate that Candida albicans activates macrophage IRE1α via C-type lectin receptor signaling independent of protein misfolding, suggesting non-canonical activation. IRE1α enhances macrophage fungicidal activity by promoting phagosome maturation, which is crucial for containing C. albicans hyphae. IRE1α facilitates early phagosomal calcium flux post-phagocytosis, which is required for phagolysosomal fusion. In macrophages lacking the IRE1α endoribonuclease domain, defective calcium flux correlates with fewer ER-early endosome contact sites, suggesting a homeostatic role for IRE1α-promoting membrane contact sites. Overall, our findings illustrate non-canonical IRE1α activation during infection and a function for IRE1α in supporting organelle contact sites to safeguard against rapidly growing microbes.

Keywords: CP: Immunology; CP: Microbiology; Candida albicans; IRE1α; calcium; fungal infection; innate immunity; phagosome.

Figure 1.. Candida albicans infection results in activation of macrophage IRE1α

(A) iBMDM cell lines (WT or IRE1^ΔR) were infected with C. albicans (Ca), treated with LPS, thapsigargin, or mock for 4 h. Xbp1 mRNA splicing was measured by semi-quantitative RT-PCR amplification of the Xbp1 transcript, followed by treatment with PstI, which recognizes a cleavage site within the 26-bp intron that is removed by IRE1α, resulting in cleavage of the unspliced isoform, specifically. (B) Immunoblot analysis of lysates from WT or IRE1^ΔR iBMDM to confirm IRE1α truncation in IRE1^ΔR cells resulting from removal of floxed exons 20 and 21. (C) Expression of spliced Xbp1 over a time course following Ca infection. (D) Expression of spliced Xbp1 at 4 h post-infection (hpi) with the indicated isolates. (E) Expression of spliced Xbp1 at 4 h following Ca infection or LPS treatment of WT or CARD9 KO iBMDM. (F) Expression of spliced Xbp1 at 4 h following Ca infection and depleted zymosan (d-zymosan) or LPS treatment of WT or IRE1^ΔR iBMDM. (G) Expression of spliced Xbp1 at 4 h following Ca infection, LPS treatment, or d-zymosan treatment of WT or TLR2/4/9 KO iBMDM. (H) Expression of spliced Xbp1 at 4 h following Ca infection or LPS treatment of two pairs of clonal iBMDM (WT or TRAF6 KO). Closed symbols are data from WT-1 and KO-1; open symbols are data from WT-2 and KO-2. Data are representative of 3–4 individual experiments. Graphs show the mean ± SEM of biological replicates (C–H). For all experiments, Ca MOI = 1, LPS treatment = 100 ng/mL, d-zymosan = 100 μg/mL, thapsigargin = 5 μM. Statistical analysis details are in STAR Methods.

Figure 2.. PRR-mediated activation of IRE1α occurs independently of misfolded protein stress

(A and B) Expression of spliced Xbp1 following C. albicans infection of iBMDM or treatment with thapsigargin (TG) as a control, compared to mock treatment. Actinomycin D (ActD; 20 μM) was used to inhibit new transcription during treatments (A), and cycloheximide (CHX; 10 μM) was used to inhibit translation during treatments (B). Relative fold changes were measured over matched mock samples. (C) Representative graphs showing fluorescence intensity of Thioflavin T (ThT) measured by flow cytometry to quantify protein misfolding in iBMDM following infection by C. albicans or treatment with LPS or thapsigargin as a positive control at 2 h. (D–F) Quantification of ThT fluorescence intensity at 2 h (D), 4 h (E), or 8 h (F) post-indicated treatment, shown as fold change over mock. (G and H) Expression of UPR-responsive genes at 4 h (G) or 6 h (H) following C. albicans infection and d-zymosan, LPS, or thapsigargin treatment. Statistical analysis details are in STAR Methods. For all experiments, Ca MOI = 1, LPS treatment = 100 ng/mL, d-zymosan = 100 μg/mL, thapsigargin = 5 μM.

Figure 3.. IRE1α promotes macrophage fungicidal activity

(A) Representative widefield micrographs of live (CFW⁺ iRFP⁺) and killed (CFW⁺ iRFP⁻) intracellular C. albicans within IRE1 WT iBMDM at 1 hpi (left) and 6 hpi (right). Scale bar, 100 μm. (B) Quantification of three independent C. albicans-killing experiments. (C) Quantification of relative C. albicans killing 6 hpi in IRE1 WT or IRE1^ΔR iBMDM treated with pharmacological inhibitors of IRE1α targeting RNase domain (MKC8866; 10 μM), kinase domain (KIRA8; 1 μM), or control DMSO treatment; relative killing is calculated as the percentage of IRE1 WT DMSO treatment. (D) Representative micrographs of C. albicans in dissociated kidney cells showing total C. albicans (anti-Candida-fluorescein isothiocyanate⁺ [FITC⁺]) in green, live C. albicans in green and magenta (anti-Candida-FITC⁺ C. albicans-expressed iRFP⁺), and CD11b⁺ cells in blue to identify host leukocytes. Images show non-myeloid-associated live and dead C. albicans (left), myeloid-associated live C. albicans (center), and myeloid-associated killed C. albicans (right). Scale bar, 10 μm. (E and F) Percentage of C. albicans killed was quantified in the kidney tissue (E) or specifically in myeloid cells (F). Graphs show the mean ± SEM of 3–4 biological replicates (B and C), or of data from individual mice (E and F). Statistical analysis details are in STAR Methods.

Figure 4.. IRE1α promotes phagosome maturation during C. albicans infection

(A) Representative images showing LAMP1 (yellow) recruitment to phagosomes containing iRFP-expressing C. albicans (magenta) in IRE1 WT or IRE1^ΔR iBMDM at indicated times post-infection. (B) Quantification of LAMP1 recruitment to phagosomes containing C. albicans in IRE1 WT or IRE1^ΔR iBMDM, as measured by LAMP1 mean fluorescence intensity associated with C. albicans-expressed iRFP. (C) Quantification of C. albicans killing in IRE1 WT and IRE1^ΔR macrophages treated with BafA or DMSO control. (D) Representative images of SRB recruitment to the phagosome containing C. albicans, indicated by white arrows, and loss of SRB association following phagosomal rupture, indicated by white asterisk. Scale bar, 10 μm. (E) Quantification of three independent experiments measuring loss of SRB from C. albicans over time in IRE1 WT or IRE1^ΔR iBMDM. Values are the mean ± SEM of 3–4 biological replicates. Statistical analysis details are in STAR Methods.

Figure 5.. IRE1α promotes phagosomal calcium flux and contact sites between the ER and endosomal network

(A) Representative micrographs of WT or IRE1^ΔR iBMDM following phagocytosis of C. albicans (20 min post-infection; MOI 2) showing early cellular calcium flux, and influx of calcium specifically in the phagosome following phagocytosis of C. albicans. Scale bar, 10 μm. (B) Quantification of calcium-high phagosomes, defined by a 1.25-fold increase of the mean fluorescence intensity of the cell (20 min post-infection; MOI 2). (C) Violin plot of the ratio of phagosomal to cytosolic mean fluorescence intensity of Fluo4 (20 min post-infection; MOI 2). (D) Quantification of LAMP1 recruitment to phagosomes containing C. albicans in IRE1 WT or IRE1^ΔR iBMDM with or without treatment of BAPTA-AM (calcium chelator), as measured by LAMP1 mean fluorescence intensity associated with C. albicans-expressed iRFP. (E) Representative confocal micrographs of PLA puncta representing sites of proximity between the ER and early endosomes/phagosomes at resting state (mock) or during C. albicans infection. Scale bar, 10 μm. (F) Quantification of PLA puncta in IRE1 WT or IRE1^ΔR iBMDM at resting state (mock). Values are the mean ± SEM from 3 to 4 biological replicates, as indicated by data points. Statistical analysis details are in STAR Methods.