Non-canonical activation of IRE1α during Candida albicans infection enhances macrophage fungicidal activity

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   Review Commons

   Aug 22, 2024

   Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

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   Reply to the reviewers

   Manuscript number:

   RC-2024-02569

   Corresponding author(s): Mary O'Riordan, Teresa O'Meara

   1. General Statements

   We thank the reviewers for their positive feedback, highlighting the significance and novelty of our work, especially regarding the novel functions of IRE1a in regulating phagosome biology during infection. We also appreciate some overarching themes that were focused on by multiple reviewers, including the role of XBP1S protein and RIDD activity, which we have addressed here. We have also added additional data, made adjustments to data presentation, and added clarifying language to address concerns from Reviewer 3. We appreciate these … More

   Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

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   Reply to the reviewers

   Manuscript number:

   RC-2024-02569

   Corresponding author(s): Mary O'Riordan, Teresa O'Meara

   1. General Statements

   We thank the reviewers for their positive feedback, highlighting the significance and novelty of our work, especially regarding the novel functions of IRE1a in regulating phagosome biology during infection. We also appreciate some overarching themes that were focused on by multiple reviewers, including the role of XBP1S protein and RIDD activity, which we have addressed here. We have also added additional data, made adjustments to data presentation, and added clarifying language to address concerns from Reviewer 3. We appreciate these constructive suggestions and include our planned experiments to address reviewer concerns here. Our specific responses to the reviewer comments are below.

   Specific figures used in the response to reviewers are in the attached file as they cannot be pasted here.

   2. Description of the planned revisions

   Reviewer 1:

   1. The demonstration of protein misfolding independent IRE1 activation should also be demonstrated using molecules such as TUDCA or 4PBA that should be innocuous regarding the splicing of XBP1s. It would also be interesting to evaluate the activation of the other arms of the UPR in particular through the phosphorylation of eIF2a, expression of ATF4 and cleavage of ATF6.

   We appreciate the suggestion to strengthen our data regarding protein misfolding-independent activation of IRE1 more robust. We note that canonical UPR transcriptional targets are not induced during C. albicans infection (Fig. 2G,H), suggesting that IRE1 is activated in the absence of a standard unfolded protein response. However, we agree that we can use additional chemical chaperones to assay this. To address this point, we will perform the suggested experiments in the presence or absence of TUDCA with C. albicans, LPS, thapsigargin, and tunicamycin. As 4PBA has been shown to inhibit protein synthesis, rather than promoting protein folding or preventing aggregation (PMC9741500), we will avoid using this compound for these assays.

   We will also perform western blots for ATF6 cleavage and eIF2a phosphorylation, although we note that eIF2a can be phosphorylated by multiple kinases and can be triggered by nutrient deprivation or changes in intracellular calcium, both of which occur during C. albicans infection (glucose: PMC6709535; calcium: data within this manuscript).

   1. The authors use thioflavin to evaluate the extent of protein misfolding. This type of stain can lead to artefactual results and in general it is rather safer to test several stainers (see for instance the work presented in PMC10720158)

   We thank the reviewer for this suggestion. We have previously tried Proteostat staining as an additional method to measure protein misfolding, but we found that it bound strongly to the C. albicans cell wall, which would result in a strong false positive signal that is not indicative of host protein misfolding (see below). Congo Red, an additional dye used in the listed reference, is also known to bind to C. albicans and perturbs cell wall synthesis (PMC266468), therefore we have avoided these dyes.

   However, to address this point, we will perform experiments utilizing poly-ubiquitin blotting, as in the suggested reference, as an orthogonal readout of protein misfolding during C. albicans infection or treatment with LPS, depleted zymosan, and thapsigargin.

   __Figure legend: Proteostat staining with ____C. albicans____ infection. __Macrophages were infected with C. albicans, and subsequently stained with Proteostat to measure protein misfolding. Proteostat bound and displayed strong fluorescence on the C. albicans cell wall.

   1. The whole study relies on the use of IRE1deltaR to impair IRE1 signaling. The authors should validate their hypothesis with an orthogonal approach, for instance with IRE1 pharmacological inhibitors (eg MKC8866 or KIRA8).

   We consider the use of genetic perturbation of IRE1 to be a strength of this manuscript, as IRE1 inhibitors have been shown to cause off-target effects (KIRA8: PMC9600248). However, to address this point, we will attempt to replicate important phenotypes, including the effect of IRE1 on calcium flux and phagolysosome fusion, using MKC8866 and KIRA8 as representative inhibitors.

   3. Description of the revisions that have already been incorporated in the transferred manuscript

   __Reviewer 1: __

   1. The authors focus on the IRE1/XBP1s signaling arm of the UPR but do not explore RIDD activity which has been linked to several infection mechanisms and lysosomal integrity (in particular by regulating the expression of BLOS1 - see PMC9119680 and PMC6446841). The authors should definitely evaluate how RIDD is activated (or not) in their experimental systems.

   We thank the reviewer for this suggestion, as we have considered potential effects of RIDD when analyzing our RNA-seq data, and are aware of the potential links between IRE1, BLOS1 (encoded by Bloc1s1) expression, and lysosome perturbations. We now add additional figures to our supplemental data (Fig. S3C-D; also shown below) showing that established RIDD targets, including Bloc1s1 are not depleted during C. albicans infection, and also not increased in IRE1 null macrophages. We add the following text to describe these findings (lines 322-326): "Additionally, we did not observe depletion of published RIDD targets (14, 65, 66) during C. albicans infection in WT macrophages (Fig. S3C; Table S2), nor increased expression of RIDD targets in IRE1ΔR macrophages, compared to IRE1 WT macrophages (Fig. S3D; Table S1.1), suggesting minimal RIDD activity during C. albicans infection." We also note that experiments with LysoSensor (Fig. 3E) suggested lysosome biogenesis is not impaired in IRE1 null macrophages. Therefore, we expect RIDD activity has negligible effects on our reported phenotypes.

   Reviewer #1 (Significance (Required)):

   The manuscript is interesting and highlights novel aspects towards the interaction between macrophages and a pathogen, candida albicans, involving the likely selective activation of IRE1. The data are novel and experimentally sound. Several controls are however missing.

   The strengths of the study are associated with the novelty of the findings, with the links that could potentially derive from this study to connect ER biology, UPR signaling and phagosome maturation

   The main weaknesses are associated i) with the fact that the authors did not evaluate RIDD activity which has already been linked with pathogen infection and with lysosome integrity, ii) with methodological aspects, in particular regarding the demonstration of the IRE1 activation independent on protein misfolding and the sole use of a genetic variant of IRE1 to test their hypotheses

   We thank Reviewer 1 for their constructive feedback and for noting the novelty of our findings. We believe that the data we have added regarding RIDD activity and our planned experiments to address additional concerns will add additional evidence to support our findings.

   Reviewer 2:

   A point that should be addressed with more detail is the correlation of fungal killing with Ca2+ fluxes and Ire1α activity, given the well-known data regarding the strong ability of the axis dectin/SYK/phospholipase Cγ to induce Ca2+ transients, a response not shared by LPS signaling, and the sequential activation of mitochondrial Ca2+ uniporter (MCU), which is a critical element of fungal killing associated with the citrate-pyruvate shuttle as a NADPH source (Seegren et al., Cell Rep. 33: 108411, 2020). Incidentally, this paper is referred in ref. 46 as a preprint, although it was accessible in Cell Reports in 2020.

   This is an excellent suggestion; we have added this topic to our discussion (lines 605-608) and have corrected the citation.

   The assay of the expression of V-ATPase complex, mitochondrial calcium uniporter, and mitochondrial uptake 1 and 2 could shed light on the dependence of fungal killing on Ire1α function.

   Thank you for this suggestion - below, we plot the transcripts comprising the V-ATPase, as well as Mcu, Micu1, and Micu2. We note that these transcripts are not perturbed in IRE1 null macrophages, suggesting that the basic functions of the V-ATPase complex and mitochondrial calcium uptake are intact in IRE1 null macrophages.

   These data are in agreement with our LysoSensor assay (Fig. 3E), which suggested that lysosome biogenesis is not impaired in IRE1 null macrophages.

   While we cannot rule out a defect in mitochondrial calcium flux from our RNA-seq data, we have added discussion around this topic to our discussion, as mentioned above.

   Expression of V-ATPase subunits and mitochondrial calcium uptake genes in C. albicans-infected IRE1 null macrophages vs C. albicans-infected IRE1 WT macrophages.

   Fig. 1A should be explained with more detail to disclose the products of PstI digestion.

   Thank you for the suggestion. We have added this information to the Figure 1 legend, "RT-PCR-amplified Xbp1 cDNA was treated with PstI, which recognizes a cleavage site within the 26 base pair intron that is removed by IRE1α activity, resulting in cleavage of the unspliced isoform, specifically."

   The anti-XBP1 antibody used to construct the blots in Fig.S1A recognizes epitopes not disclosed by the manufacturers, but they have to pertain to the N-terminal peptide sequence shared by sXBP1 and uXBP1. Showing full lanes encompassing both protein isoforms would allow a better appraisal of protein expression. In connection to point 4, the use of an antibody reactive to the epitopes expressed in sXBP1 in cell lysates or, preferentially in nuclear fractions, could be most valuable to rule out the dependence of the effect of Ire1α on the trans-activating function of sXBP1.

   We have un-cropped these westerns and now show spliced and unspliced XBP1 products on a single image in Fig. S1A.

   On page 23, the mention to Fig. 5A should be changed to Fig. 5B.

   We have fixed this mis-labeling, thank you for calling this to our attention.

   Line 209. I understand gene synthesis refers to gene expression.

   We have clarified this in the text, thank you for the suggestion.

   Line 394. What is the reason to study the cytokine-signature of Candida in LPS-primed cells?

   Thank you for the question; we have added the following text (lines 413-414) to clarify that LPS is used for inflammasome priming:

   "Therefore, we tested secretion of IL-1β, TNF, and IL-6 from WT and IRE1ΔR macrophages after LPS treatment to transcriptionally prime the NLRP3 inflammasome components, followed by C. albicans infection (Fig. 5D-F)."

   Numerous studies have shown that C. albicans can trigger macrophage pyroptosis, resulting in production of pro-inflammatory cytokines like IL-1b, which can also be influenced by phagosome rupture (PMC3910967). However, this requires inflammasome transcriptional priming, and LPS is commonly used to prime macrophages for inflammasome activation in vitro. Therefore, we perform a short pre-treatment with LPS for NLRP3 inflammasome priming to subsequently measure its activation following C. albicans infection, using secreted cytokines as a readout. We also note that macrophages in vivo may not be naive and are often M1-polarized by the microbial or cytokine environment, thus inflammasome priming is likely common during in vivo infection.

   Reviewer #2 (Significance (Required)):

   This study focuses on an aspect not usually addressed in papers devoted to the UPR.

   If more data are shown as suggested, the paper could be of interest for a wider audience

   We thank the reviewer for their positive feedback about the novelty of our work and agree that the suggested experiments will bolster our data and story.

   ---

   Reviewer 3:

   Fig. 2:

   Panel A-B: same question as for Fig. 1. The variation in TG DMSO-induced splicing is huge. The effects of the treatments with CHX or Act D are smaller than the variation between experiments with TG DMSO alone. As long as that variation is not controlled for, it is impossible to draw any conclusion from the inhibitors. In this regard, it is very difficult to interpret data if they are not done in one and the same experiment.

   The variability in thapsigargin fold change over mock likely represents differences in basal Xbp1 expression. We consistently see complete Xbp1 splicing in response to thapsigargin treatment (see Fig. 1A). Additionally, we note that thapsigargin treatment is used only as a positive control, not as a physiologically relevant treatment, as it results in unmitigated ER stress that triggers cell death (PMC6986015).

   We have removed the following sentence, *"Translation inhibition using cycloheximide was sufficient to alleviate Xbp1 splicing specifically in response to thapsigargin, likely by reducing the nascent protein folding burden (Fig. 2B)," *since our data are plotted on separate graphs, matched to their respective controls, for appropriate comparisons.

   4. Description of analyses that authors prefer not to carry out

   ---

   Reviewer 1:

   1. Since the IRE1/XBP1 arm of the UPR is also involved in lipid biosynthesis which might be required for phagosome maturation, the authors should perform XBP1s rescues in IRE1 deficient cells to ensure that their observation is XBP1s dependent or IRE1 dependent.

   As we do not see XBP1S protein induced in wild-type macrophages at any timepoint during our C. albicans infection scheme (Fig. S1A-B), we interpret our results as being XBP1S-independent. If we were to add back XBP1S with constitutive expression, we would be overexpressing the protein relative to C. albicans infected wild-type macrophages (in which we do not see measurable XBP1S expression). Therefore, we believe these experiments would not address a physiologically-relevant scenario.

   1. The authors should evaluate in what compartment IRE1 is activated upon CA infection, does that happen in the ER or in the ER fraction fused to phagosomes?

   This is an interesting question for future exploration. In order to answer this question with existing tools, we would need to perform biochemical fractionation of infected cells to isolate an ER-phagosome contact site fraction, followed by phos-tag gel analysis of IRE1 activation in the ER fraction, compared to the ER-phagosome contact site fraction. However, a biochemical fractionation protocol to distinguish the ER fraction from ER-phagosome contact sites has not yet been developed, to our knowledge, and we believe it is outside the scope of this study to develop such a technique.

   We have added additional text regarding this intriguing question to our discussion (lines 549-553).

   Reviewer 2:

   ---

   Infection at a MOI 1 of C. albicans is a ratio of infecting agent/susceptible targets not very high for a non-soluble stimulus with limited diffusion in the culture medium. Although I recognize the difficulty of quantitating adhered cell, the mention to 80% confluence makes it more difficult the appraisal of the actual MOI. The delayed time-course of Xbp splicing under these conditions can be explained by the time required for in vitro proliferation, Candida damage, and diffusion of fungal patterns. A study with viable Candida at MOI 5 in human monocyte-derived dendritic cells, which show a robust capacity for non-opsonic phagocytosis associated with C-type lectin receptors only showed initial hypha formation after 2 hours (Rodriguez et al., J. Biol. Chem. 289, P22942-22957, 2014). Consistent with the requirement of a time lag for infecting agent to attain levels of expression consistent with a net response, 16 hours have been considered an appropriate time-course to assay sXBP1 expression following SARS-CoV2 infection (Fernandez et al., Biochim Biophys Acta Mol Basis Dis. 1870(5):167193, 2024). I wonder if a higher MOI could show a similar kinetics.

   We use lower MOI in part due to the size and ability of C. albicans to undergo extensive hyphal growth if its numbers greatly exceed the number of host cells. From our microscopy data, we can see that C. albicans spreads well throughout the culture plate (see Fig. 3A, Fig. 4A). We and others have observed considerable death of macrophage cultures after 12 hours with Candida infection, even at low MOI (PMC6709535), therefore we avoid later timepoints in these assays and all other in vitro assays in our manuscript.

   As all of our in vitro experiments are performed within an 8 hour window of infection, whether XBP1S is induced at later timepoints by C. albicans or depleted zymosan would not alter the conclusions of the rest of our results.

   sXBP1 can be present in nuclear fractions in resting cells, which suggests the involvement of post-translational modifications for the display of transcriptional activity.

   As we do not see induction of XBP1S in our lysates after C. albicans infection, it is unlikely that post-translational modification is influencing its function, although we agree post-translational modification is a likely regulatory control over XBP1S during the unfolded protein response.

   The independence of sXBP1 transcriptional activity from canonical UPR associated with misfolded protein stress is well known from the seminal paper by Martinon et al., (ref.6). Moreover, the expression of CHOP, the final effector of the PERK route, encoded by DDIT3 gene, has been found to be blunted by Candida (Rodriguez et al., J. Biol. Chem. 289, P22942-22957,2014). This is additional evidence for the recruitment of sXBP1 transcriptional activity in the absence of canonical UPR.

   As mentioned, we found that XBP1S protein is not induced during C. albicans infection at any timepoint in our experiments (Fig. S1A-B). Importantly, the work referenced by the reviewer uses RAW267.7 cells, which (as mentioned by the authors) constitutively express CHOP as a result of Abel leukemia virus infection. Based on this specific overexpression, we believe this phenotype is not comparable to our bone marrow-derived macrophages.

   Reviewer 3:

   Fig. 1:

   Panel 1C: please remove outlier in 4h timepoint. This implies that the experiment needs to be redone to reduce variation

   We have performed an outlier test on these data, which revealed that this data point is not a statistical outlier, therefore we do not feel that its removal is appropriate (see below).

   Panel 1E-H: how is the splicing efficiency determined and normalized? How to explain the big differences in splicing efficiency of Xbp1 upon LPS stimulation (appr. 4 to 6 times in E, G and H versus 30-fold in panel F). Where does this difference come from?

   Panel H, outlier needs to be removed.

   We do occasionally see differences in magnitude of Xbp1 splicing in different cell lines or experiments, especially with controls, which may be caused by differences in the basal level of Xbp1 expression, especially as Xbp1 levels have been shown to be affected by circadian rhythm in certain cell types (PMCID: PMC11214543; PMCID: PMC6959563).

   In panel H, an outlier test reveals that these are not statistical outliers, therefore we feel their removal is inappropriate as we do not wish to mask biological variation. Moreover, this graph includes two cell lines (open and closed circles), showing that our data are robust across multiple independent cell lines and are an appropriate measure of experimental replicates.

   Fig. 2:

   Panel A-B: same question as for Fig. 1. The variation in TG DMSO-induced splicing is huge. The effects of the treatments with CHX or Act D are smaller than the variation between experiments with TG DMSO alone. As long as that variation is not controlled for, it is impossible to draw any conclusion from the inhibitors. In this regard, it is very difficult to interpret data if they are not done in one and the same experiment.

   The variability in thapsigargin fold change over mock likely represents differences in basal Xbp1 expression. We consistently see complete Xbp1 splicing in response to thapsigargin treatment (see Fig. 1A). Additionally, we note that thapsigargin treatment is used only as a positive control, not as a physiologically relevant treatment, as it results in unmitigated ER stress that triggers cell death (PMC6986015).

   We have removed the following sentence, "Translation inhibition using cycloheximide was sufficient to alleviate Xbp1 splicing specifically in response to thapsigargin, likely by reducing the nascent protein folding burden (Fig. 2B), since our data are plotted on separate graphs, matched to their respective controls, for appropriate comparisons.

   Below, we plot all data together with replicate matching, although our major interpretation of these data is that C. albicans infection can trigger Xbp1 splicing with or without new gene expression, and not about the impact of the inhibitors on the control treatment thapsigargin.

   Please provide a scheme of how the experiment was performed, at what time were the inhibitors provided, at what time point the inducers? What are matched mock samples. Which mock samples were chosen since they differ from one experiment to the next? Please plot all the data for one and the same experiment in one graph so that the reader can easily compare the results of DMSO, DMSO + inducer, DMSO + inducer + inhibitor. Indicate whether the points in the graph are technical or experimental repetitions.

   -How to explain the increase in XBP1 splicing in combination with ActD? Was this due to differences in Gapdh expression? Where did the authors control for cell death? Please provide the data.

   Below is a scheme of the experimental treatments. We have now clarified in the figure legend that inhibitors (ActD and CHX) are added at the same time as experimental treatments (Mock, Ca, TG). All data included in the original submission are biological replicates, as stated in the figure legend. We have now re-written the figure legend to clearly indicate that these are biological replicates.

   All data are normalized such that the effects of the drugs are directly compared (for example, the fold change over Mock for Candida is matched to its drug treatment; Mock DMSO vs Ca DMSO and Mock ActD vs Ca ActD, or Mock CHX vs Ca CHX). Actinomycin D does inhibit new transcription, although IRE1 can cleave existing Xbp1 transcript. We now show conditions normalized to DMSO Mock in Supplemental Figure 2, which allows visualization of the effects of ActD and CHX on Xbp1-S abundance in comparison to control DMSO treatment, while also seeing the relative changes in Xbp1 splicing caused by C. albicans or thapsigargin treatment (see below).

   -Is RT-qPCR a reliable readout when actinomycinD is used? How can new genes be transcribed.

   We interpret RT-qPCR data as a readout of transcript abundance, rather than transcription. Therefore, we are not measuring new gene expression here, but whether the existing Xbp1 transcript can be cleaved by IRE1. Based on the technique, we can still measure changes in Xbp1-S abundance.

   Panel D: where is TG at 4h and 6h?

   We do not include thapsigargin at later timepoints to avoid autofluorescence from excessive cell death. We include thapsigargin as a positive control at the early 2h timepoint, but note that LPS is sufficient to increase thioflavin T intensity at the 8h timepoint.

   Panel G, why was Ddit3 included here as this is not a typical IRE1 dependent gene (rather PERK dependent). What about IRE1 specific genes such as Sec61 or Sec24a?

   We have added additional text (lines 235-240; "Finally, we measured induction of UPR-responsive genes by RT-qPCR in response to C. albicans infection, LPS and depleted zymosan treatment, or thapsigargin treatment, to further test whether IRE1α activation occurs without canonical UPR induction (Fig. 2G-H). C. albicans infection and depleted zymosan treatment did not lead to induction of UPR-responsive genes (Ddit3, Grp78, Grp94, and total Xbp1) at 4 or 6 hours.") to clarify that the purpose of this figure is to add evidence that IRE1 activation is independent of the canonical UPR response (indicating that IRE1 is likely specifically activated independently of the other UPR branches) during C. albicans infection. Therefore, the transcripts measured are canonical UPR-responsive transcripts, rather than IRE1/XBP1S targets (although some are overlapping).

   Below are RNA-seq data comparing Sec61a1, Sec61a2, and Sec24a in IRE1 null macrophages, compared to IRE1 WT macrophages. While there is less expression of Sec61a1 in IRE1 null macrophages, Sec61a2 and Sec24a are largely unaffected. These data support our finding that XBP1S protein is not induced during* C. albicans* infection.

   Did the authors also check for RIDD activity?

   As mentioned above in response to Reviewer 1, we now add additional figures to our supplemental data (Fig. SX; also shown below) showing that established RIDD targets are not depleted during C. albicans infection in WT macrophages, and also not increased in IRE1 null macrophages. Therefore, we expect RIDD activity has negligible effects on our reported phenotypes.

   Fig. 3:

   Panel C and D look convincing. Lamp1 is a well-known RIDD target gene (see Osorio et al., Nat Imm, 2014). Did the authors check Lamp1 expression in presence and absence of IRE1 and could RIDD explain their phenotype?

   As shown above, Lamp1 transcript expression is not strongly perturbed in IRE1 null macrophages. If RIDD activity were depleting Lamp1 transcript abundance, we would expect to see increased* Lamp1* expression in IRE1 null macrophages. We also note that our experiments using LysoSensor (Fig. 3E) suggested that lysosome biogenesis is not impaired, but more specifically, lysosome recruitment to the phagosome is impaired in IRE1 null macrophages.

   Fig. 4, but especially Fig 5 and Fig 6 suffer from very bad imaging quality. Both Fig 5A and Fig 6A are completely uninterpretable. The SRB staining is all over the cells and it is totally unclear how the authors interpret this as phagosomal leakage or not. Fig. 6A is even worse and appears nothing but vague background. It is difficult to understand how the authors make graphs based on these types of images and dare to draw any conclusions.

   In Figure 4, we observe some photobleaching from frequent image acquisition, which is necessary to capture calcium flux dynamics. Image brightness across the timecourse is adjusted in the same way such that we do not attempt to hide the effects of photobleaching. However, our analyses account for photobleaching over time, and the phagosomal calcium flux is clear and quantifiable. `

   In Figure 5, the sulforhodamine B pulse-chase assay involves loading of the endosomal system with SRB, thus the cells are expected to ingest a considerable amount of SRB and it will distribute throughout the endosomal network. However, as endosomes fuse, we also observe fusion with the C. albicans-containing phagosome and SRB will surround C. albicans hyphae. Our analysis pipeline first segments C. albicans hyphae (see below) and measures SRB signal in proximity to the phagosome. Thus, we measure loss of phagosome-associated SRB over time, as C. albicans ruptures the phagosome, in hundreds of macrophages. This is a standard assay that has been previously used for this purpose (PMID: 33022213; PMID: 30131363).

   For Figure 6, we have added additional wide-field images that we believe will clarify how these images can be readily quantified (Fig. 6A, shown below). The purpose of the previous Fig. 6A (now Fig. 6B) is to demonstrate single cell examples of live and dead C. albicans using the dual-fluorescence assay, although we quantify much wider fields for sufficient numbers. We hope the amended figures provide additional clarity.

   Fig. 7 is again an example where differences in expression are mainly due to one or a few complete outliers, and it is hard to understand why the authors did not repeat these experiments to reduce the problems in variation to get proper data sets before submission.

   After performing outlier tests, we have found a total of 4 data points that are statistical outliers from all of the panels in Figure 7. These included the highest data point in each genotype in the female IL-1Ra levels (Fig. 7A, second graph), the highest data point among the male IRE1 fl/fl mice IL-1Ra levels (Fig. 7B, second graph), and the highest data point among the male TNF levels in IRE1 fl/fl + LysM-Cre mice (Fig. 7B, third graph). We have removed these data points in our updated graphs and changed the text to only point out differences in serum TNF and IL-6 levels. Moreover, our interpretation includes that serum cytokine levels are not different in male mice. However, no other data points are statistical outliers, therefore we believe their removal is inappropriate.

   While the paper started nicely and showed an interesting hypothesis (Fig. 3), the remaining part of the paper was of very poor quality and was not ready for submission.

   We thank the reviewer for the constructive feedback and believe that the addition of data and clarifications we have added will demonstrate that our data are of sufficient quality to support our conclusions.

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   Referee #3

   Evidence, reproducibility and clarity

   Reviewer comments for "Non-canonical activation of IRE1a by Candida albicans infection promotes macrophage phagosomal calcium flux to enhance fungal killing"

   This paper describes a role for IRE1 in controlling Candida albicans (Ca) infection in macrophages. The authors show that Ca infection slightly induces IRE1 activity as monitored by XBP1 splicing, however this does not result in XBP1 protein expression, nor in IRE1-dependent target gene expression. The authors propose that IRE1 controls phagosomal maturation, as measured by defects in LAMP1 recruitment to Ca containing phagosomes. This would be due to a defect in calcium flux … More

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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   ---

   Referee #3

   Evidence, reproducibility and clarity

   Reviewer comments for "Non-canonical activation of IRE1a by Candida albicans infection promotes macrophage phagosomal calcium flux to enhance fungal killing"

   This paper describes a role for IRE1 in controlling Candida albicans (Ca) infection in macrophages. The authors show that Ca infection slightly induces IRE1 activity as monitored by XBP1 splicing, however this does not result in XBP1 protein expression, nor in IRE1-dependent target gene expression. The authors propose that IRE1 controls phagosomal maturation, as measured by defects in LAMP1 recruitment to Ca containing phagosomes. This would be due to a defect in calcium flux at the phagosomal level leading to an increased propensity to rupture and cytosolic escape of the pathogen. While the data are interesting and the defect in LAMP1 recruitment to the phagosome convincing, the majority of the data are difficult to interpret due to the poor quality. This concerns specifically all imaging experiments but also the ELISAs and qPCRs where differences are due to the effect of outliers rather than to the behavior of a complete population. Therefore, most experiments need to be redone and complemented with additional approaches before any firm conclusions can be drawn. Specific details and examples are provided below.

   Fig. 1:

   Panel 1C: please remove outlier in 4h timepoint. This implies that the experiment needs to be redone to reduce variation

   Panel 1E-H: how is the splicing efficiency determined and normalized? How to explain the big differences in splicing efficiency of Xbp1 upon LPS stimulation (appr. 4 to 6 times in E, G and H versus 30-fold in panel F). Where does this difference come from?

   Panel H, outlier needs to be removed.

   Fig. 2:

   Panel A-B: same question as for Fig. 1. The variation in TG DMSO-induced splicing is huge. The effects of the treatments with CHX or Act D are smaller than the variation between experiments with TG DMSO alone. As long as that variation is not controlled for, it is impossible to draw any conclusion from the inhibitors. In this regard, it is very difficult to interpret data if they are not done in one and the same experiment. Please provide a scheme of how the experiment was performed, at what time were the inhibitors provided, at what time point the inducers? What are matched mock samples. Which mock samples were chosen since they differ from one experiment to the next? Please plot all the data for one and the same experiment in one graph so that the reader can easily compare the results of DMSO, DMSO + inducer, DMSO + inducer + inhibitor. Indicate whether the points in the graph are technical or experimental repetitions.

   • How to explain the increase in XBP1 splicing in combination with ActD? Was this due to differences in Gapdh expression? Where did the authors control for cell death? Please provide the data.
   • Is RT-qPCR a reliable readout when actinomycinD is used? How can new genes be transcribed.

   Panel D: where is TG at 4h and 6h?

   Panel G, why was Ddit3 included here as this is not a typical IRE1 dependent gene (rather PERK dependent). What about IRE1 specific genes such as Sec61 or Sec24a?

   Did the authors also check for RIDD activity?

   Fig. 3:

   Panel C and D look convincing. Lamp1 is a well-known RIDD target gene (see Osorio et al., Nat Imm, 2014). Did the authors check Lamp1 expression in presence and absence of IRE1 and could RIDD explain their phenotype?

   Fig. 4, but especially Fig 5 and Fig 6 suffer from very bad imaging quality. Both Fig 5A and Fig 6A are completely uninterpretable. The SRB staining is all over the cells and it is totally unclear how the authors interpret this as phagosomal leakage or not. Fig. 6A is even worse and appears nothing but vague background. It is difficult to understand how the authors make graphs based on these types of images and dare to draw any conclusions.

   Fig. 7 is again an example where differences in expression are mainly due to one or a few complete outliers, and it is hard to understand why the authors did not repeat these experiments to reduce the problems in variation to get proper data sets before submission.

   While the paper started nicely and showed an interesting hypothesis (Fig. 3), the remaining part of the paper was of very poor quality and was not ready for submission.

   Significance

   The study presents interesting hypothesis but unfortunately the data are not of sufficient quality

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   Aug 22, 2024

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   Referee #2

   Evidence, reproducibility and clarity

   This an interesting report in a widely explored area. This makes it necessary to pigeonhole the new data provided by the study. The paper addresses two issues encompassing a scope distinct from studies focusing on the cytokine-signature and the role of sXbp1. This research singles out fungal killing and Ire1α versus sXbp1 function. More precisely, the reduction of cytokine expression in Ire1fl/fl LysMCre as compared to WT discloses an opposing function of Ire1α and its target sXbp1 in cytokine expression that requires mechanistic explanation.

   1. A point that should be addressed with more detail is the correlation of fungal killing with …

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   Referee #2

   Evidence, reproducibility and clarity

   This an interesting report in a widely explored area. This makes it necessary to pigeonhole the new data provided by the study. The paper addresses two issues encompassing a scope distinct from studies focusing on the cytokine-signature and the role of sXbp1. This research singles out fungal killing and Ire1α versus sXbp1 function. More precisely, the reduction of cytokine expression in Ire1fl/fl LysMCre as compared to WT discloses an opposing function of Ire1α and its target sXbp1 in cytokine expression that requires mechanistic explanation.

   1. A point that should be addressed with more detail is the correlation of fungal killing with Ca2+ fluxes and Ire1α activity, given the well-known data regarding the strong ability of the axis dectin/SYK/phospholipase Cγ to induce Ca2+ transients, a response not shared by LPS signaling, and the sequential activation of mitochondrial Ca2+ uniporter (MCU), which is a critical element of fungal killing associated with the citrate-pyruvate shuttle as a NADPH source (Seegren et al., Cell Rep. 33: 108411, 2020). Incidentally, this paper is referred in ref. 46 as a preprint, although it was accessible in Cell Reports in 2020.
   2. The assay of the expression of V-ATPase complex, mitochondrial calcium uniporter, and mitochondrial uptake 1 and 2 could shed light on the dependence of fungal killing on Ire1α function.
   3. Infection at a MOI 1 of C. albicans is a ratio of infecting agent/susceptible targets not very high for a non-soluble stimulus with limited diffusion in the culture medium. Although I recognize the difficulty of quantitating adhered cell, the mention to 80% confluence makes it more difficult the appraisal of the actual MOI. The delayed time-course of Xbp splicing under these conditions can be explained by the time required for in vitro proliferation, Candida damage, and diffusion of fungal patterns. A study with viable Candida at MOI 5 in human monocyte-derived dendritic cells, which show a robust capacity for non-opsonic phagocytosis associated with C-type lectin receptors only showed initial hypha formation after 2 hours (Rodriguez et al., J. Biol. Chem. 289, P22942-22957, 2014). Consistent with the requirement of a time lag for infecting agent to attain levels of expression consistent with a net response, 16 hours have been considered an appropriate time-course to assay sXBP1 expression following SARS-CoV2 infection (Fernandez et al., Biochim Biophys Acta Mol Basis Dis. 1870(5):167193, 2024). I wonder if a higher MOI could show a similar kinetics.
   4. Fig. 1A should be explained with more detail to disclose the products of PstI digestion.
   5. sXBP1 can be present in nuclear fractions in resting cells, which suggests the involvement of post-translational modifications for the display of transcriptional activity.
   6. The independence of sXBP1 transcriptional activity from canonical UPR associated with misfolded protein stress is well known from the seminal paper by Martinon et al., (ref.6). Moreover, the expression of CHOP, the final effector of the PERK route, encoded by DDIT3 gene, has been found to be blunted by Candida (Rodriguez et al., J. Biol. Chem. 289, P22942-22957,2014). This is additional evidence for the recruitment of sXBP1 transcriptional activity in the absence of canonical UPR.
   7. The anti-XBP1 antibody used to construct the blots in Fig.S1A recognizes epitopes not disclosed by the manufacturers, but they have to pertain to the N-terminal peptide sequence shared by sXBP1 and uXBP1. Showing full lanes encompassing both protein isoforms would allow a better appraisal of protein expression. In connection to point 4, the use of an antibody reactive to the epitopes expressed in sXBP1 in cell lysates or, preferentially in nuclear fractions, could be most valuable to rule out the dependence of the effect of Ire1α on the trans-activating function of sXBP1.
   8. On page 23, the mention to Fig. 5A should be changed to Fig. 5B.
   9. Line 209. I understand gene synthesis refers to gene expression.
   10. Line 394. What is the reason to study the cytokine-signature of Candida in LPS-primed cells?

   Significance

   This study focuses on an aspect not usually addressed in papers devoted to the UPR. If more data are shown as suggested, the paper could be of interest for a wider audience

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4. 

   Review Commons

   Aug 22, 2024

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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   Referee #1

   Evidence, reproducibility and clarity

   The authors show that in macrophages, IRE1 activation (independent of improperly folded proteins) is essential to promote fungicidal activity towards candida albicans (CA) in vitro and in vivo by ensuring phagosome maturation through the preservation of calcium fluxes

   Major comments

   1. The demonstration of protein misfolding independent IRE1 activation should also be demonstrated using molecules such as TUDCA or 4PBA that should be innocuous regarding the splicing of XBP1s. It would also be interesting to evaluate the activation of the other arms of the UPR in particular through the phosphorylation of eIF2a, expression of ATF4 and …

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   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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   Referee #1

   Evidence, reproducibility and clarity

   The authors show that in macrophages, IRE1 activation (independent of improperly folded proteins) is essential to promote fungicidal activity towards candida albicans (CA) in vitro and in vivo by ensuring phagosome maturation through the preservation of calcium fluxes

   Major comments

   1. The demonstration of protein misfolding independent IRE1 activation should also be demonstrated using molecules such as TUDCA or 4PBA that should be innocuous regarding the splicing of XBP1s. It would also be interesting to evaluate the activation of the other arms of the UPR in particular through the phosphorylation of eIF2a, expression of ATF4 and cleavage of ATF6.
   2. Since the IRE1/XBP1 arm of the UPR is also involved in lipid biosynthesis which might be required for phagosome maturation, the authors should perform XBP1s rescues in IRE1 deficient cells to ensure that their observation is XBP1s dependent or IRE1 dependent.
   3. The authors use thioflavin to evaluate the extend of protein misfolding. This type of stain can lead to artefactual results and in general it is rather safer to test several stainers (see for instance the work presented in PMC10720158)
   4. The authors should evaluate in what compartment IRE1 is activated upon CA infection, does that happen in the ER or in the ER fraction fused to phagosomes?
   5. The authors focus on the IRE1/XBP1s signaling arm of the UPR but do not explore RIDD activity which has been linked to several infection mechanisms and lysosomal integrity (in particular by regulating the expression of BLOS1 - see PMC9119680 and PMC6446841). The authors should definitely evaluate how RIDD is activated (or not) in their experimental systems.
   6. The whole study relies on the use of IRE1deltaR to impair IRE1 signaling. The authors should validate their hypothesis with an orthogonal approach, for instance with IRE1 pharmacological inhibitors (eg MKC8866 or KIRA8).

   Significance

   The manuscript is interesting and highligths novel aspects towards the interaction between marcrophages and a pathogen, candida albicans, involving the likely selective activation of IRE1. The data are novel and experimentally sound. Several controls are however missing.

   The strengths of the study are associated with the novelty of the findings, with the links that could potentially derive from this study to connect ER biology, UPR signaling and phagosome maturation

   The main weaknesses are associated i) with the fact that the authors did not evaluate RIDD activity which has already been linked with pathogen infection and with lysosome integrity, ii) with methodological aspects, in particular regarding the demonstration of the IRE1 activation independent on protein misfolding and the sole use of a genetic variant of IRE1 to test their hypotheses

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5. 

   Version published to 10.1101/2023.10.02.560560v2 on bioRxiv

   May 2, 2024
6. 

   Version published to 10.1101/2023.10.02.560560v1 on bioRxiv

   Oct 2, 2023