Systemic Lupus Erythematosus Serum Stimulation of Human Intestinal Organoids Induces Changes in Goblet Cell Differentiation and Mitochondrial Fitness

  1. Inga Viktoria Hensel
  2. Szabolcs Éliás
  3. Michelle Steinhauer
  4. Bilgenaz Stoll
  5. Salvatore Benfatto
  6. Wolfgang Merkt
  7. Stefan Krienke
  8. Hanns-Martin Lorenz
  9. Jürgen Haas
  10. Brigitte Wildeman
  11. Martin Resnik-Docampo

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Abstract

Human intestinal epithelial cells are the interface between potentially harmful luminal content and basally residing immune cells. Their role is not only nutrient absorption but also the formation of a tight monolayer that constantly secrets mucus creating a multi-layered protective barrier. Alterations in this barrier can lead to increased gut permeability which is frequently seen in individuals with chronic extraintestinal autoimmune diseases, such as Systemic Lupus Erythematosus (SLE). Despite recent advances in identifying alterations in gut microbiota composition in SLE patients, not much attention has been given to the epithelial barrier itself. To date, it remains largely unexplored which role and function intestinal epithelial cells have in SLE pathology. Here, we present a unique near-physiologic in vitro model specifically designed to examine the effects of SLE on the epithelial cells. We utilize human colon organoids that are stimulated with serum obtained from SLE patients. Combining bulk and scRNA transcriptomic analysis with functional assays revealed that SLE serum stimulation induced a unique expression profile marked by a type I interferon gene signature. Additionally, organoids exhibited decreased mitochondrial fitness, alterations in mucus composition and imbalanced cellular composition. Similarly, transcriptomic analysis of SLE human colon biopsies revealed a downregulation of epithelial secretory markers. Our work uncovers a crucial connection between SLE and intestinal homeostasis that might be promoted in vivo through the blood, offering insights into the causal connection of barrier dysfunction and autoimmune diseases.

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    Reply to the reviewers

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    Authors used organoid technology to study the effects of the serum from lupus patients on intestinal epithelium. By culturing organoids derived from human colon crypts, they specifically determined the response of epithelial cells to inflammatory mediators present in lupus serum. Using bulk and scRNA-seq, authors found that secretory cells function and differentiation were impaired as well as the mitochondrial metabolism. These effects were shown to be mediated by type 1 interferon in combination with other pro-inflammatory cytokines present in lupus serum.

    The reduction of mucus secretion after SLE-serum treatment and the downregulation of tight junctions' genes seem to indicate an increased permeability of the epithelial barrier, thus it would be interesting to determine the expression and distribution of tight junction proteins and to test in the organoids whether the paracellular permeability is increased upon SLE-serum treatment. These analyses will give a functional result of this in vitro model.

    If the organoids take a few days to culture and the material is available, the measurement of paracellular permeability may take no more than 2 weeks. It is true that they will need a microneedle to inject the FITC-Dextran 4K into the organoids and record the images for 24h.

    • We thank the reviewer for suggesting this experiment. Reviewer #2 had the same suggestion. The results obtained elevate our overall results and the quality of our research.
    • Tight junction protein expression is cell-type dependent as shown in literature1,2 and in our scRNA-seq analysis (Suppl. Table 7). Additionally, we observed that the expression changes of ZO-1(TJP1; Fig. 7A) are accumulated in colonocytes. That might be the reason we could not detect significant changes in organoid sections with staining for tight junction proteins ZO-1 and Occludin and analyzing all cell types. The data of the permeability assay however was able to show that the function of the tight junctions is altered. If this change is caused by changes in protein abundance as suggested by reported transcriptomic changes remains to be elucidated in future research using single-cell proteomic approaches.
    • For the permeability assay we stimulated organoid monolayers for 72h with SLE or control serum. The measured translocation of FITC showed an increased barrier leakiness in SLE compared to control condition. These functional results not only support our findings presented in our study that the epithelial barrier is altered upon SLE serum stimulation, but they also provide the definitive proof of concept highlighting the crucial connection between SLE and intestinal barrier integrity. Furthermore, it shows that changes on transcriptional level and alterations in cell type composition translate into a barrier dysfunction which could have potentially detrimental effects in vivo. The data has been added to Figure 1H, line 722-731; 746-751.

    I would like to know which of the donor's cells were used from figure 2 on and why.

    • Organoid line I was used for the 24h stimulation and for the 72h stimulation whereas organoid line II was used for the 72h stimulation only. Due to limitations in serum availability we had to limit the experiments that exceeded the initial transcriptomic analysis to one organoid line. After confirming that the serum was affecting both organoid lines, we continued using organoid line II.

    The bioinformatics analyses using gene expression data and scRNASeq were well done. No comments.

    Reviewer #1 (Significance (Required)):

    For the field of autoimmunity, to study the crosstalk between the systemic response and the gut epithelium response results quite important as the increased permeability of the gut epithelial barrier has been suggested to fuel the systemic inflammation in lupus. However, as the author mention, there is not enough information about the interaction of epithelial cells and the systemic inflammatory mediators in lupus. This system can be useful to determine a personalized treatment for patients by testing the effect of individual serum on organoids. Moreover, the use of organoids can be extended to study the gut epithelium response in other autoimmune diseases mediated by type 1 interferon.

    Increased permeability of the gut epithelial barrier has been related with lupus development. In humans, it is not known whether it is a cause or consequence, but in lupus mouse models it has been demonstrated that there is a reduction of the systemic autoimmune response concomitant with a reduction of gut permeability. The authors have validated an in vitro model that can be used to study how gut epithelium is affected by systemic inflammatory mediators and that will help to develop novel therapeutic approaches or personalize treatments.

    Thank you for your insightful comments. We are encouraged to see that the reviewer has accurately grasped the core purpose and implications of our research. The intricate relationship between gut epithelial barrier permeability and lupus development is indeed vital for expanding our scientific knowledge and for future therapeutic breakthroughs. We are happy that the overall message we aimed to convey with our research was well captured and appreciated by the reviewer. We believe our in vitro model will serve as a foundation for further detailed studies and for the development of therapeutic strategies in this field. Your acknowledgment of our work inspires us to persist in our research efforts.

    Interest stakeholders: Clinical and basic researchers in autoimmunity, gastroenterology, and rheumatology.

    My field of expertise is systemic and organ-specific autoimmunity at cellular and molecular level. My work covers autoimmunity and gut microbiota. I study how B cells regulate the microbiota composition and how that microbiota impacts gut permeability and inflammation in mouse lupus models. On the other hand, the bioinformatics analyses are well-done for both bulk RNASeq and scRNASeq.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    The group of Dr. Resnik-Docampo provides a very elegant study on two patient lines for SLE. The study is definitely very interesting and opens many scientific avenues that are worthy of being explored further. Major comments: -Barrier integrity or its alterations can be tested in organoids with specific dyes, I feel this would give definitive proof of concept.

    • We thank the reviewer for the suggestion. Reviewer #1 had the same suggestion. The results obtained elevate our overall results and the quality of our research.
    • For the permeability assay we stimulated organoid monolayers for 72h with SLE or control serum. The measured translocation of FITC showed an increased barrier leakiness in SLE compared to control condition. These functional results not only support our findings presented in our study that the epithelial barrier is altered upon SLE serum stimulation, but they also provide the definitive proof of concept highlighting the crucial connection between SLE and intestinal barrier integrity. Furthermore, it shows that changes on transcriptional level and alterations in cell type composition translate into a barrier dysfunction which could have potentially detrimental effects in vivo. The data has been added to Figure 1H, line 722-731; 746-751.

    -In supplementary figure 1 a caspase 3 staining is presented, please show a positive control for caspase 3 staining on organoids or alternstively use a different method to prove no differential cell death.

    • Due to the processing of the organoids it is difficult to have a positive control. However, we can confirm that the used cleaved Caspase 3 antibody is able to detect cell death by the following staining. There are positive cells in the center of the organoid where dead cells accumulate. Additionally in Figure 1C we show with a cytotoxicity assay measuring LDH release that there is no significant difference between both groups. Furthermore, brightfield imaging showed no obvious differences and the DEGs show also no evidence of increased cell death.

    -Serum from SLE patients reduces drastically Edu positivity, it would be interesting to see a clonogenicity assay to see whether this reflects on reduced stem cell clonogenic potential

    • We agree. However, this analysis goes beyond the time and material limitations we have in our project.

    -Goblet cells in the colon are very heterogeneous, which subpopulations of goblet cell are reduced? how does this affect mucus composition?

    • We see that subpopulation GC4 is significantly reduced upon SLE serum stimulation (Fig. 7C and Suppl. Fig. 7C). Overall, we see a trend of a general reduction of all GC subpopulation and an indication that there might be also a shift in subtype abundance. We would need to increase the study population to be able to draw any conclusions on how exactly the GC subpopulations change. So far not much is known about how the different GC subpopulations affect mucus composition. This field is completely understudied especially in the human intestine. Future projects should focus on mucus composition and how it is changed with changes of the subpopulations (even under physiological conditions).
    • We thank the reviewer for the question about the mucus composition. We included the analysis of FCGBP protein abundance (Fig. 4C and Suppl. Fig. 4D; line 245-247) in our study. The increased FCGBP protein abundance upon SLE serum stimulation which is in line with our transcriptomic changes complements our data and supports our hypothesis that the mucus composition is altered. This new data improves the quality of our research.

    Minor comments:

    • Please provide an hypothesis on how mitochondrial alterations are linked to altered lineage progeny of stem cells. This should be discussed more in depth.
    • It is known that cells in the crypt compartment that undergo rapid division depend on glycolysis for ATP production once the cell differentiates it switches to oxidative phosphorylation.3 In vitro it has been shown that differentiation coincides with the switch to oxidative phosphorylation.4 There is a complex interplay between Notch signaling and FoxO transcription factors which is driving differentiation and cell fate decision.5 Especially the differentiation towards the secretory lineage highly depends on the metabolic switch from glycolysis to oxidative phosphorylation. Furthermore, even mucus secretion itself is dependent on oxidative phosphorylation.6 This is in line with our differentiation experiment where less oxidative phosphorylation coincided with the absence of goblet cells. We hypothesize that the changed cellular composition upon SLE serum stimulation is at least partly reflected in the altered mitochondrial function. If the decrease in the secretory lineage alone can explain the seen mitochondrial changes needs to be further elucidated. While a detailed examination at single-cell level analysis of mitochondrial function might be able to answer if they drive the altered cell differentiation, such an in-depth analysis is beyond the scope of this article and would be best addressed in a dedicated study on the topic. Within the scope of our analysis and considering the still limited knowledge of mitochondrial function in specific cell types of the human colon we included some more discussion (line 696-698 and 702-703).

    -Many antimicrobial peptides are changed, this could reflect on microbiome composition as well as mucus composition and properties, which I am sure will be the topic of future studies. This should be discussed more in depth.

    • We hypothesize that the alterations in antimicrobial peptide expression along with the seen changes in major mucus components would be translated into changes in mucus composition. Since the mucus serves as the niche for gut microbiota this could lead to changes in microbiome composition. It is of high interest to analyze the mucus composition of the stimulated organoids. Furthermore, assays analyzing the killing capacity of the potentially secreted antimicrobial peptides could help us to understand the relevance of the observed changes. We addressed this in line 714-715 and line 750-752.

    Reviewer #2 (Significance (Required)):

    The study is useful for both broad and specialized audiences. The findings are interesting and of relevance to the field of SLE, gut epithelial biology. The strength of the manuscript is that it opens many scientific avenues, its weakness is that they are not mechanistically dissected to the fullest rendering the study a bit descriptive. Nonetheless, I consider positively the manuscript after a minor revision given the major message of the paper can be proven.

    Thank you for your constructive feedback. We are genuinely encouraged by your recognition of the utility and relevance of our study for both broad and specialized audiences in the fields of SLE and gut epithelial biology. Your acknowledgment resonates with our larger objectives: beyond merely exploring the specific connection between SLE and intestinal leakiness, our aim has been to create a methodological approach that illuminates novel avenues to study complex diseases. It is deeply heartening to realize that this overarching message and intent were clearly understood and agreed upon by the reviewer. We recognize and appreciate your insights into the strengths and areas for improvement of our manuscript, and we are committed to addressing the highlighted points to further enhance our contribution to the field.

    __ Reviewer #3 (Evidence, reproducibility and clarity (Required)):__

    Inga Viktoria Hensel et al. used colon organoid to study the impact of lupus patients' serum on gut epithelial barrier. The exposure of SLE serum on colon organoids increased gene expression related to cell cycle, chromosome organization, mitochondrial function as well as interferon signaling, but downregulated that related to secretion, cytoskeleton, and anchoring junctions of the cells. Higher type I IFN in the SLE serum and unregulated interferon signature genes post stimulation suggest a potential role of type I interferon in this process. The addition of a type 1 interferon receptor (IFNAR1) antagonist, Anifrolumab, blocked the stimulation function of SLE serum but the combination of IFN-2α and control serum failed to recapitulate the results from SLE serum, suggesting that more than one cytokine was involved. SLE serum exposure altered metabolic profiles of organoids with a significant increase of basal respiration and ATP production. Stimulating organoid with SLE serum confirmed an alteration in cell differentiation with a loss of secretory lineage. scRNA-seq analysis revealed that colon organoid had all major cell types from colon in vivo. SLE serum stimulation shifted cell differentiation with decreased number of goblet cells and downregulated mucin, AMP and other components that were required for gut barrier integrity.

    Finally, the authors performed a gene expression analysis of colon biopsies derived from SLE patients and healthy controls. While the authors should be commended to attempt a validation of the results obtained with organoids, the small sample size and patient heterogeneity prevented a statistical analysis. Some genes involved in absorption and ion transport as well as secretory lineage showed a similar trend with organoid assay, suggesting that colon organoids may be a good tool for future studies. However, it is noticeable that the biopsies from SLE patients did not show the IFN signature and the decreased in Muc2 expression, which dominated the gene signature of organoids exposed to SLE serum. There is no information about the disease activity of the SLE patients, as well as their IFN activity, which makes difficult to interpret these results.

    • We thank the reviewer for the questions and suggestions. They helped us to improve our manuscript and make it more concise for the reader.

    Specific concerns:

    1. Line 107, Why did The authors use 72 hours post treatment. Are other timepoints available and have similar results?
    • We used two different time points, 24h and 72h. The nature of the organoid culture limits the total length of the experiment. Organoids can be cultured for a maximum of 5-7 days. Differentiation from the stem cell to the fully differentiated cell takes 5-7 days. Preliminary results showed that serum stimulation prior to day 2 led to a decrease in organoid survival, most likely because serum stimulation in general induces differentiation. We therefore chose 72h as the stimulation that mimics a chronic exposure as a differentiating cell would face in vivo. With this time span we were able to see manifestations in cell differentiation changes but avoided beginning cell death to prolonged culture. However, we also wanted to understand a more acute exposure to the serum. Therefore, we chose 24h as a second stimulation duration. With this time point we were able to detect initial changes in cell fate decision which was important in the interpretation of the accumulated effects seen after 72h.

    Figure 1D, how do the authors explain the heterogeneity among SLE samples (2, 3, 4, 5) on organoid line II? These samples do not seem to correlate with cytokine levels shown in Fig. 2. This issue may be worth exploring further, such as correlation between cytokine levels and gene expression.

    • The heterogeneity seen among the SLE samples most likely reflects the complex composition of the serum itself. A similar heterogeneity (PC1 axis) is also seen for the controls. The epithelial cells eventually will react to all contained factors showing an integrated response that makes it difficult to correlate to a single cytokine.

    Line 144, the 2 outlier SLE serum samples are not same between organoid lines with NO. 1&5 in Organoid line II and with NO. 1&4 in Organoid line I. The statement is misleading.

    • We corrected the statement according to the suggestions (Line 146-148).

    Line 169, IFN-a2 and IL-6 are not significantly different.

    • The statement was rephrased (Line 166-174).

    Line 179-180, Reduced fitness of organoids exposed to SLE serum is an overstatement. It was not directly tested, and there is no difference in apoptosis.

    • The term ‘reduced fitness’ referred to the results seen in the mitochondrial stress test. We rephrased the parts to make the statement more concise (Line 182).

    Line 242-243: SLE serum stimulation induced MUC2 high expression in Organoid II but lower level in organoid I (Figure 4B & Figure S4C). This is a major discrepancy that needs to be addressed.

    • This discrepancy comes from the different differentiation dynamics observed in both organoid lines. Therefore, for the downstream analysis we considered the results from both lines to have a robust analysis. With the results from the 24h timepoint and the scRNA-seq which were performed with either of the lineages respectively, we can be certain that the overall seen effect on the secretory lineage is a valid finding (we addressed this in line 229-232).

    Line 251-252: How do authors make sure that "we were facing an effect on the differentiation process rather than cell type loss"?

    • We can exclude an increase of cell death since we did not see changes in LDH release (Fig. 1C) and cleaved Caspase 3 abundance (Suppl. Fig. 1A) when we compared both conditions. Additionally, the data from the 24h stimulation time point showed the reduction of transcription factors (Fig. 4J) important for differentiation which manifested in a reduction of secretory cell markers upon longer stimulation (72h) (edited statement in line 256-258).

    Line 259, How about apoptosis gene levels here?

    • Apoptosis markers BAX and BCL2 are not amongst the differentially expressed genes. (see Suppl. Table 4).

    Line 290 : It has been shown that the response of colon explants to IFN-α was variable among donors (https://www.sciencedirect.com/science/article/pii/S2352345X16301084#undfig1). This study should be cited. Was the response to IFNa tested on both organoids?

    • The reference was included (Line 669-671). The response to IFNα was only tested in organoid line II given the limited availability of the serum that was used for co-stimulation. Overall, however, we saw an effect in both donor lines and less response was rather connected to the serum, not the organoid donor. In the publication reported interindividual heterogeneity depends on the therewith connected release of IL-18. Future studies could include analysis of cytokine release from the organoids after serum stimulation and a higher number of organoid lines to validate our findings.

    Line 305-307: Is single cell sequencing from single organoid line or from combined? Do two organoid lines show different distributions?

    • scRNA-seq was performed using organoid line II. Due to limited resources, we unfortunately could not include another organoid line.

    Reviewer #3 (Significance (Required)):

    While there is mounting evidence of an altered intestinal barrier integrity in SLE patients, there is little insights in the mechanisms. Using colon organoids is a novel approach with great potentials to investigate this issue. The strongest signature found by the authors was type IFN, which is indicates that the colon epithelial cells respond in a similar manner to other cell types. The secretory and absorption genes are of potential greater interest to unraveling mechanisms to gut alteration in SLE. This study is of interest for audiences interested in lupus basic and clinic research, as well as investigators working of gut barrier integrity.

    Thank you for your constructive feedback. We are encouraged by your acknowledgment of our novel approach using colon organoids to explore the altered intestinal barrier integrity in SLE patients. Your emphasis on the significance of the type IFN signature and the importance of secretory and absorption genes aligns with our perspective.

    Incorporating the intestinal barrier functional experiment emphasizes the translational nature of our study. We agree with your view that our findings are relevant for those involved in both basic and clinical lupus research, as well as specialists in intestinal biology. Your encouraging feedback strengthens our conviction in the wider significance of our work. We are motivated to keep bridging the gap between these two essential areas of study.

    References

    1. Pearce, S. C. et al. Marked differences in tight junction composition and macromolecular permeability among different intestinal cell types. BMC Biol. 16, 1–16 (2018).
    2. Kishida, K., Pearce, S. C., Yu, S., Gao, N. & Ferraris, R. P. Nutrient sensing by absorptive and secretory progenies of small intestinal stem cells. Am. J. Physiol. Liver Physiol. 312, G592–G605 (2017).
    3. Rath, E., Moschetta, A. & Haller, D. Mitochondrial function — gatekeeper of intestinal epithelial cell homeostasis. Nat. Rev. Gastroenterol. Hepatol. 15, 497–516 (2018).
    4. Rodríguez-Colman, M. J. et al. Interplay between metabolic identities in the intestinal crypt supports stem cell function. Nature 543, 424–427 (2017).
    5. Ludikhuize, M. C. et al. Mitochondria Define Intestinal Stem Cell Differentiation Downstream of a FOXO/Notch Axis. Cell Metab. 32, 889-900.e7 (2020).
    6. Sünderhauf, A. et al. Loss of Mucosal p32/gC1qR/HABP1 Triggers Energy Deficiency and Impairs Goblet Cell Differentiation in Ulcerative Colitis. Cmgh 12, 229–250 (2021).
  2. Review Commons

    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

    Inga Viktoria Hensel et al. used colon organoid to study the impact of lupus patients' serum on gut epithelial barrier. The exposure of SLE serum on colon organoids increased gene expression related to cell cycle, chromosome organization, mitochondrial function as well as interferon signaling, but downregulated that related to secretion, cytoskeleton, and anchoring junctions of the cells. Higher type I IFN in the SLE serum and unregulated interferon signature genes post stimulation suggest a potential role of type I interferon in this process. The addition of a type 1 interferon receptor (IFNAR1) antagonist, Anifrolumab, blocked the stimulation function of SLE serum but the combination of IFN-2α and control serum failed to recapitulate the results from SLE serum, suggesting that more than one cytokine was involved. SLE serum exposure altered metabolic profiles of organoids with a significant increase of basal respiration and ATP production. Stimulating organoid with SLE serum confirmed an alteration in cell differentiation with a loss of secretory lineage. scRNA-seq analysis revealed that colon organoid had all major cell types from colon in vivo. SLE serum stimulation shifted cell differentiation with decreased number of goblet cells and downregulated mucin, AMP and other components that were required for gut barrier integrity.

    Finally, the authors performed a gene expression analysis of colon biopsies derived from SLE patients and healthy controls. While the authors should be commended to attempt a validation of the results obtained with organoids, the small sample size and patient heterogeneity prevented a statistical analysis. Some genes involved in absorption and ion transport as well as secretory lineage showed a similar trend with organoid assay, suggesting that colon organoids may be a good tool for future studies. However, it is noticeable that the biopsies from SLE patients did not show the IFN signature and the decreased in Muc2 expression, which dominated the gene signature of organoids exposed to SLE serum. There is no information about the disease activity of the SLE patients, as well as their IFN activity, which makes difficult to interpret these results.

    Specific concerns:

    1. Line 107, Why did The authors use 72 hours post treatment. Are other timepoints available and have similar results?
    2. Figure 1D, how do the authors explain the heterogeneity among SLE samples (2, 3, 4, 5) on organoid line II? These samples do not seem to correlate with cytokine levels shown in Fig. 2. This issue may be worth exploring further, such as correlation between cytokine levels and gene expression.
    3. Line 144, the 2 outlier SLE serum samples are not same between organoid lines with NO. 1&5 in Organoid line II and with NO. 1&4 in Organoid line I. The statement is misleading.
    4. Line 169, IFN-a2 and IL-6 are not significantly different.
    5. Line 179-180, Reduced fitness of organoids exposed to SLE serum is an overstatement. It was not directly tested, and there is no difference in apoptosis.
    6. Line 242-243: SLE serum stimulation induced MUC2 high expression in Organoid II but lower level in organoid I (Figure 4B & Figure S4C). This is a major discrepancy that needs to be addressed.
    7. Line 251-252: How do authors make sure that "we were facing an effect on the differentiation process rather than cell type loss"?
    8. Line 259, How about apoptosis gene levels here?
    9. Line 290 : It has been shown that the response of colon explants to IFN-α was variable among donors (https://www.sciencedirect.com/science/article/pii/S2352345X16301084#undfig1). This study should be cited. Was the response to IFNa tested on both organoids?
    10. Line 305-307: Is single cell sequencing from single organoid line or from combined? Do two organoid lines show different distributions?

    Significance

    While there is mounting evidence of an altered intestinal barrier integrity in SLE patients, there is little insights in the mechanisms. Using colon organoids is a novel approach with great potentials to investigate this issue. The strongest signature found by the authors was type IFN, which is indicates that the colon epithelial cells respond in a similar manner to other cell types. The secretory and absorption genes are of potential greater interest to unraveling mechanisms to gut alteration in SLE. This study is of interest for audiences interested in lupus basic and clinic research, as well as investigators working of gut barrier integrity.

  3. Review Commons

    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 #2

    Evidence, reproducibility and clarity

    The group of Dr. Resnik-Docampo provides a very elegant study on two patient lines for SLE. The study is definitely very interesting and opens many scientific avenues that are worthy of being explored further.

    Major comments:

    • Barrier integrity or its alterations can be tested in organoids with specific dyes, I feel this would give definitive proof of concept.
    • In supplementary figure 1 a caspase 3 staining is presented, please show a positive control for caspase 3 staining on organoids or alternstively use a different method to prove no differential cell death.
    • Serum from SLE patients reduces drastically Edu positivity, it would be interesting to see a clonogenicity assay to see whether this reflects on reduced stem cell clonogenic potential
    • Goblet cells in the colon are very heterogeneous, which subpopulations of goblet cell are reduced? how does this affect mucus composition?

    Minor comments:

    • Please provide an hypothesis on how mitochondrial alterations are linked to altered lineage progeny of stem cells. This should be discussed more in depth.
    • Many antimicrobial peptides are changed, this could reflect on microbiome composition as well as mucus composition and properties, which I am sure will be the topic of future studies. This should be discussed more in depth.

    Significance

    The study is useful for both broad and specialised audiences. The findings are interesting and of relevance to the field of SLE, gut epithelial biology. The strength of the manuscript is that it opens many scientific avenues, its weakness is that they are not mechanistically dissected to the fullest rendering the study a bit descriptive. Nonetheless, I consider positively the manuscript after a minor revision given the major message of the paper can be proven.

  4. Review Commons

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

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    Authors used organoid technology to study the effects of the serum from lupus patients on intestinal epithelium. By culturing organoids derived from human colon crypts, they specifically determined the response of epithelial cells to inflammatory mediators present in lupus serum. Using bulk and scRNA-seq, authors found that secretory cells function and differentiation were impaired as well as the mitochondrial metabolism. These effects were shown to be mediated by type 1 interferon in combination with other pro-inflammatory cytokines present in lupus serum.

    The reduction of mucus secretion after SLE-serum treatment and the downregulation of tight junctions' genes seem to indicate an increased permeability of the epithelial barrier, thus it would be interesting to determine the expression and distribution of tight junction proteins and to test in the organoids whether the paracellular permeability is increased upon SLE-serum treatment. These analyses will give a functional result of this in vitro model.

    If the organoids take a few days to culture and the material is available, the measurement of paracellular permeability may take no more than 2 weeks. It is true that they will need a microneedle to inject the FITC-Dextran 4K into the organoids and record the images for 24h.

    I would like to know which of the donor's cells were used from figure 2 on and why.

    The bioinformatics analyses using gene expression data and scRNASeq were well done. No comments.

    Significance

    For the field of autoimmunity, to study the crosstalk between the systemic response and the gut epithelium response results quite important as the increased permeability of the gut epithelial barrier has been suggested to fuel the systemic inflammation in lupus. However, as the author mention, there is not enough information about the interaction of epithelial cells and the systemic inflammatory mediators in lupus. This system can be useful to determine a personalized treatment for patients by testing the effect of individual serum on organoids. Moreover, the use of organoids can be extended to study the gut epithelium response in other autoimmune diseases mediated by type 1 interferon.

    Increased permeability of the gut epithelial barrier has been related with lupus development. In humans, it is not known whether it is a cause or consequence, but in lupus mouse models it has been demonstrated that there is a reduction of the systemic autoimmune response concomitant with a reduction of gut permeability. The authors have validated an in vitro model that can be used to study how gut epithelium is affected by systemic inflammatory mediators and that will help to develop novel therapeutic approaches or personalize treatments.

    Interest stakeholders: Clinical and basic researchers in autoimmunity, gastroenterology, and rheumatology.

    My field of expertise is systemic and organ-specific autoimmunity at cellular and molecular level. My work covers autoimmunity and gut microbiota. I study how B cells regulate the microbiota composition and how that microbiota impacts gut permeability and inflammation in mouse lupus models. On the other hand, the bioinformatics analyses are well-done for both bulk RNASeq and scRNASeq.

  5. Version published to 10.1101/2023.07.04.547690v2 on bioRxiv
  6. Version published to 10.1101/2023.07.04.547690v1 on bioRxiv