Selective Complement Inhibition in Anti-p200 Pemphigoid: Immune Infiltrate Profiles and Therapeutic Implications Compared to Bullous Pemphigoid

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Autoimmune bullous diseases, especially pemphigoids, are in the focus of skin diseases because of their increasing prevalence in the elderly population of the developed countries. Drug-induced forms and other atypical forms often have nonbullous clinical manifestation and atypical immunohistological results which make the diagnosis delayed and more difficult. The therapy of pemphigoid is usually not complicated, but in anti-p200 pemphigoid there can be higher proportion of patients who do not respond well to the conventional corticosteroid therapy or show higher rate of relapses. Among others, this is the main reason to understand the pathomechanism of the disease and find new therapeutic strategies. 

 

The investigation clearly showed the histological presentation of anti-p200 pemphigoid and analyzed the inflammatory infiltrates in lesional and perilesional skin. In inflammatory infiltrates there were predominantly neutrophil cells and most of them expressed complement receptors C5aR1 and/or C5aR2, this expression was higher in lesional skin. Complement-fixating activity was also investigated in anti-p200 pemphigoid and in bullous pemphigoid, interestingly they have found stronger C3-fixating capacity in BP autoantibodies. In my opinion, the small number of BP patients compared to the anti-p200 patients may have resulted inadequate results. The in vitro use of selective complement inhibitors showed promising results in both anti-p200 and bullous pemphigoid. The latter means the use of selective complement inhibitors can be new targeted therapeutic options in BP, too. 

 

Our comments and questions are:

1. In the manuscript, they have mentioned that they had 11 patients with anti-p200 pemphigoid whose skin samples were taken and used for staining. In my opinion, it would be also necessary to describe in the methods how the exact diagnosis was made in these patients, because anti-p200 pemphigoid cannot be differentiated from BP or EBA based on the direct immunofluorescent staining or salt split skin test.
2. For complement fixation test they have used serum from 50 anti-p200 patients. These 50 patients are not mentioned in the Methods section. 50 patients in such a rare disease are respectable number. How long did it take to collect this amount of serum? It should be mentioned in the first part of the Methods.
3. How can they explain the low rate of serum inducing complement fixation in anti-p200 patients? It is true that only few data are available in the literature but based on the references it should be higher in anti-p200 patients, similarly in bullous pemphigoid.

The main question, is there any role of complement activation in anti-p200 pemphigoid, has been answered. The manuscript is clear, presented in a well-structured manner. The topic is relevant, they have used appropriate investigational methods, and the results are demonstrated in a proper manner with excellent immunofluorescent images. The results clearly support the hypothesis. References are appropriate.

In my opinion, the manuscript is acceptable for publication with minor revision (based on the above-mentioned suggestions).

Author Response

In the manuscript, they have mentioned that they had 11 patients with anti-p200 pemphigoid whose skin samples were taken and used for staining. In my opinion, it would be also necessary to describe in the methods how the exact diagnosis was made in these patients, because anti-p200 pemphigoid cannot be differentiated from BP or EBA based on the direct immunofluorescent staining or salt split skin test.

Response: We thank the reviewer for this important comment. The Methods section (Section 2.1) has now updated to clarify how the diagnosis of anti-p200 pemphigoid was established as follows:

“In brief, anti-p200 pemphigoid was characterized by: (i) tense blisters on normal and ery-thematous skin; (ii) linear deposits of IgG and⁄or C3 at the dermal-epidermal junction detected by direct IF microscopy; (iii) binding of IgG deposition along the dermal side of the split by indirect IF microscopy on 1 mol L-1 NaCl-split normal human skin; (iv) reactivity of serum IgG antibodies with a 200-kDa protein by Western blotting using extracts generated from normal human dermis, and (v) ELISA employing a recombinant monomeric C-terminal fragment of human laminin c1 (hLAMC1-cterm).”

For complement fixation test they have used serum from 50 anti-p200 patients. These 50 patients are not mentioned in the Methods section. 50 patients in such a rare disease are respectable number. How long did it take to collect this amount of serum? It should be mentioned in the first part of the Methods.

Response: We thank the reviewer for this comment. The inclusion of sera from 50 patients with anti-p200 pemphigoid has now clarified in the Methods section (Section 2.1). Owing to the rarity of the disease, these 50 serum samples were collected over an extended period, from 2008 to 2017, at the Department of Dermatology, Schleswig-Holstein University Hospital.

How can they explain the low rate of serum inducing complement fixation in anti-p200 patients? It is true that only few data are available in the literature but based on the references it should be higher in anti-p200 patients, similarly in bullous pemphigoid.

Response: We thank the reviewer for this insightful comment. To our knowledge, no published studies have directly compared serum-mediated complement fixation between anti-p200 pemphigoid and BP. Available data for anti-p200 pemphigoid are limited to small case series reporting C3 deposition by DIF rather than standardized CFT. In our study, CFT demonstrated BMZ C3c deposition in 40% (20/50) of anti-p200 pemphigoid sera versus 87% (13/15) of BP sera, representing the first head-to-head quantification under identical experimental conditions. This difference is consistent with trends observed in DIF studies, in which C3 deposition is reported slightly more frequently in BP than in anti-p200 pemphigoid.

The lower CFT positivity in anti-p200 pemphigoid may reflect disease heterogeneity, differences in circulating autoantibody titers, and/or variability in epitope specificity (e.g., laminin β4 versus laminin γ1), which can influence complement-fixing capacity. Additionally, complement activation in anti-p200 pemphigoid may be more localized to lesional skin, limiting detectable serum-mediated activity in vitro. Importantly, our findings demonstrate that anti-p200 autoantibodies can activate complement in a substantial subset of patients, supporting a pathogenic role for complement while highlighting interindividual variability in effector mechanisms.

The Discussion section has now been revised accordingly:

“We assessed the complement-fixating potential of anti-p200 pemphigoid IgG using the CFT and examined pharmacological blockade of autoantibody-induced complement activation in vitro. CFT is a well-established assay long applied to the study of BP and pemphigoid gestationis [31]. In this assay, complement-inactivated patient sera were incubated with healthy human skin cryosections. Normal human hirudin plasma was then added as a complement source, with or without specific inhibitors. Among the 50 anti-p200 pemphigoid sera, 20 (40%) induced BMZ complement fixation, compared with 13 of 15 (87%) BP sera, indicating a stronger C3-fixating capacity of BP autoantibodies. This difference is consistent with trends observed in DIF studies, in which C3 deposition is reported slightly more frequently in BP than in anti-p200 pemphigoid [12, 23, 30]. Both cohorts could be classified as strongly, mildly, or non-complement-fixing subgroups. This heterogeneity, more pronounced in anti-p200 pemphigoid, supports a pathogenic role for complement while highlighting interindividual variability in effector mechanisms. It also suggests that CFT could potentially identify complement-dependent subsets suitable for targeted therapy. Since sera were collected at initial diagnosis during active disease before systemic immunosuppression, the observed variability is unlikely to be attributable to treatment effects or disease chronicity. Whether complement-fixing capacity correlates with disease severity, mucosal involvement, or epitope specificity (e.g., laminin β4 vs. laminin γ1) remains unknown and warrants investigations in future longitudinal studies.”

​The main question, is there any role of complement activation in anti-p200 pemphigoid, has been answered. The manuscript is clear, presented in a well-structured manner. The topic is relevant, they have used appropriate investigational methods, and the results are demonstrated in a proper manner with excellent immunofluorescent images. The results clearly support the hypothesis. References are appropriate.

In my opinion, the manuscript is acceptable for publication with minor revision (based on the above-mentioned suggestions).

Response: We thank the reviewer for the constructive comments and positive feedback on our manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

General Statement

The scientific report enclosed by Emtenani et al. encloses a well-designed and comprehensive study addressing the role of complement activation in anti-p200 pemphigoid, with a comparative analysis to bullous pemphigoid (BP). It is clearly written, methodologically rigorous, and highly relevant to the field of autoimmune blistering diseases. In my humble opinion, the data are convincing and provide valuable mechanistic insights, particularly in terms of inflammatory infiltrate composition and the potential of pathway-specific complement inhibition as a therapeutic strategy.

Overall, the manuscript represents a meaningful contribution to the literature and is suitable for publication after minor revision as commented below:

1. Clinical Contextualization of Complement Fixation Findings

The complement fixation test (CFT) data are clearly presented and reveal interesting heterogeneity among anti-p200 pemphigoid patients. It would further strengthen the manuscript if the authors could briefly clarify whether sera were obtained during active disease or different disease stages.

2. Methodological Clarifications

I am asking myself how representative fields were selected for quantification of immune cell infiltrates. Please describe this in the text for the potential reader.

Please indicate whether quantification of IHC & IF staining was performed in a blinded manner. It would be helpful for the readers as well.

3. Figures & Legends

Figures 4 & 5 are highly informative (congratulations!) – in my eyes, minor simplification of the legends OR clearer labeling of the complement components analyzed (C3 vs. C5) could further improve readability.

• Please specify the number of independent experiments used for semi-quantitative analyses in the figure legends.

 4. Terminology and Presentation

• Ensure consistent terminology for anti-p200 pemphigoid throughout the manuscript.

Namely, in some sections (especially „Results“ and „Discussion“), phrases like
“anti-p200 pemphigoid patients”, “anti-p200 pemphigoid sera”, or simply “anti-p200 pemphigoid” are alternated with more implicit wording such as: “the anti-p200 cohort”, “these patients”, “this disease”.

Please find the compromise for this wording & statements throughout the manuscript.

5. Discussion and Perspective

The Discussion is well-balanced and comprehensive. Nevertheless, a brief concluding sentence highlighting how these findings may inform future diagnostic stratification or therapeutic studies in anti-p200 pemphigoid could further strengthen the translational perspective.

6. Language

The manuscript is generally very well written; only minor editorial polishing is suggested to improve readability in a few longer sentences – as pointed out below:

• Results (page 5, line 172)

“While the average total number of C5aR1⁺ and/or C5aR2⁺ cells was higher in anti-p200 pemphigoid lesions compared with perilesional skin, the percentage of C5aR1⁺ and/or C5aR2⁺ neutrophils in lesional skin was slightly lower than in perilesional skin.”

Remark & Suggestion:

This sentence contains two contrasting observations and multiple qualifiers, making it dense. The sentence could be split into two for improved readability, separating absolute cell numbers from proportional differences.

 

• Discussion (pages 10, line 246)

“Complement deposition has been demonstrated by direct IF of perilesional skin and by immunoperoxidase staining of FFPE lesional tissue from BP patients, suggesting that complement activation contributes to lesion formation.”

Remark & Suggestion:

This is scientifically sound but slightly long. It could be tightened by:

• Removing “has been demonstrated by”
• Or splitting methods and interpretation into two shorter clauses

 

• Discussion (page 10, line 263)

“In this assay, complement-inactivated patient sera were incubated with healthy human skin cryosections, followed by the addition of normal human hirudin plasma as a complement source, with or without specific inhibitors.”

Remark & Suggestion:

The sentence is methodologically dense and includes several sequential steps. It could be shortened or divided to improve readability, particularly for readers less familiar with the CFT methodology.

 

• Discussion (page 10, line 269)

“This heterogeneity suggests that CFT may identify complement-dependent subgroups amenable to targeted therapy.”

Remark & Suggestion:

This sentence is concise but conceptually rich. I suggest to add a brief qualifier (e.g., “potentially”) Or slightly simplifying “complement-dependent subgroups amenable to targeted therapy”. This would soften the claim and improve stylistic flow.

 

• Conclusion (page 12, line 324)

“The distinct complement-fixating capacities of autoantibodies in anti-p200 pemphigoid compared with BP suggest disease-specific complement activation signatures and potentially distinct therapeutic thresholds.”

Remark & Suggestion:

This sentence contains multiple layered concepts. Consider splitting this sentence into two to clearly distinguish mechanistic insight from therapeutic implications.

 

My General Recommendation: Minor revision

The authors are to be congratulated on a carefully conducted and thoughtfully presented study. Addressing the points above would further enhance clarity and translational impact, but no additional experiments are required.

Author Response

Overall, the manuscript represents a meaningful contribution to the literature and is suitable for publication after minor revision as commented below:

1. Clinical Contextualization of Complement Fixation Findings

The complement fixation test (CFT) data are clearly presented and reveal interesting heterogeneity among anti-p200 pemphigoid patients. It would further strengthen the manuscript if the authors could briefly clarify whether sera were obtained during active disease or different disease stages.

Response: We thank the reviewer for this insightful comment. Serum samples were collected at the time of initial diagnosis from anti-p200 pemphigoid patients, prior to the initiation of any systemic immunosuppressive therapy, corresponding to active disease. Accordingly, the observed heterogeneity in complement-fixating capacity cannot be attributed to treatment effects or differences in disease chronicity.

The Discussion section has now been revised accordingly:

“We assessed the complement-fixating potential of anti-p200 pemphigoid IgG using the CFT and examined pharmacological blockade of autoantibody-induced complement activation in vitro. CFT is a well-established assay long applied to the study of BP and pemphigoid gestationis [31]. In this assay, complement-inactivated patient sera were incubated with healthy human skin cryosections. Normal human hirudin plasma was then added as a complement source, with or without specific inhibitors. Among the 50 anti-p200 pemphigoid sera, 20 (40%) induced BMZ complement fixation, compared with 13 of 15 (87%) BP sera, indicating a stronger C3-fixating capacity of BP autoantibodies. This difference is consistent with trends observed in DIF studies, in which C3 deposition is re-ported slightly more frequently in BP than in anti-p200 pemphigoid [12, 23, 30]. Both cohorts could be classified as strongly, mildly, or non-complement-fixing subgroups. This heterogeneity, more pronounced in anti-p200 pemphigoid, supports a pathogenic role for complement while highlighting interindividual variability in effector mechanisms. It also suggests that CFT could potentially identify complement-dependent subsets suitable for targeted therapy. Since sera were collected at initial diagnosis during active disease before systemic immunosuppression, the observed variability is unlikely to be attributable to treatment effects or disease chronicity. Whether complement-fixing capacity correlates with disease severity, mucosal involvement, or epitope specificity (e.g., laminin β4 vs. laminin γ1) remains unknown and warrants investigations in future longitudinal studies.”

 

2. Methodological Clarifications

I am asking myself how representative fields were selected for quantification of immune cell infiltrates. Please describe this in the text for the potential reader.

Response: We thank the reviewer for this comment. For quantification of inflammatory infiltrates, cells were manually counted in two representative fields per lesional and perilesional section for each patient, preferably one from the right side and one from the left side of the section. This information has now been added to the Methods section (Section 2.2).

Please indicate whether quantification of IHC & IF staining was performed in a blinded manner. It would be helpful for the readers as well.

Response: All quantification of IHC & IF staining was performed in a blinded manner. This has now been added to the Methods section (Section 2.2 and 2.3).

 

3. Figures & Legends

Figures 4 & 5 are highly informative (congratulations!) – in my eyes, minor simplification of the legends OR clearer labeling of the complement components analyzed (C3 vs. C5) could further improve readability. Please specify the number of independent experiments used for semi-quantitative analyses in the figure legends.

Response: We thank the reviewer for this point. Minor adjustments have been made to Figures 4 and 5 and their legends to improve readability, including clearer labeling of the complement components analyzed (C3 vs. C5). Data from three independent experiments were used for semi-quantitative analysis, and this has now been added to the figure legends.

 

4. Terminology and Presentation

Ensure consistent terminology for anti-p200 pemphigoid throughout the manuscript.

Namely, in some sections (especially „Results“ and „Discussion“), phrases like “anti-p200 pemphigoid patients”, “anti-p200 pemphigoid sera”, or simply “anti-p200 pemphigoid” are alternated with more implicit wording such as: “the anti-p200 cohort”, “these patients”, “this disease”. Please find the compromise for this wording & statements throughout the manuscript.

Response: Consistent terminology has now been applied throughout the manuscript.

5. Discussion and Perspective

The Discussion is well-balanced and comprehensive. Nevertheless, a brief concluding sentence highlighting how these findings may inform future diagnostic stratification or therapeutic studies in anti-p200 pemphigoid could further strengthen the translational perspective.

Response: We thank the reviewer for this interesting comment. A concluding statement highlighting the translational relevance has now been added to the Discussion section:

“Taken together, these findings highlight the potential of complement-fixation profiling, alongside routine diagnostic tools, to stratify anti-p200 pemphigoid patients and guide future diagnostic and complement-targeted therapeutic studies.”

6. Language

The manuscript is generally very well written; only minor editorial polishing is suggested to improve readability in a few longer sentences – as pointed out below:

Results (page 5, line 172): “While the average total number of C5aR1⁺ and/or C5aR2⁺ cells was higher in anti-p200 pemphigoid lesions compared with perilesional skin, the percentage of C5aR1⁺ and/or C5aR2⁺ neutrophils in lesional skin was slightly lower than in perilesional skin.”

Remark & Suggestion: This sentence contains two contrasting observations and multiple qualifiers, making it dense. The sentence could be split into two for improved readability, separating absolute cell numbers from proportional differences.

Discussion (pages 10, line 246): “Complement deposition has been demonstrated by direct IF of perilesional skin and by immunoperoxidase staining of FFPE lesional tissue from BP patients, suggesting that complement activation contributes to lesion formation.”

Remark & Suggestion: This is scientifically sound but slightly long. It could be tightened by: Removing “has been demonstrated by” Or splitting methods and interpretation into two shorter clauses.

Discussion (page 10, line 263): “In this assay, complement-inactivated patient sera were incubated with healthy human skin cryosections, followed by the addition of normal human hirudin plasma as a complement source, with or without specific inhibitors.”

Remark & Suggestion: The sentence is methodologically dense and includes several sequential steps. It could be shortened or divided to improve readability, particularly for readers less familiar with the CFT methodology.

Discussion (page 10, line 269): “This heterogeneity suggests that CFT may identify complement-dependent subgroups amenable to targeted therapy.”

Remark & Suggestion: This sentence is concise but conceptually rich. I suggest to add a brief qualifier (e.g., “potentially”) Or slightly simplifying “complement-dependent subgroups amenable to targeted therapy”. This would soften the claim and improve stylistic flow.

Conclusion (page 12, line 324): “The distinct complement-fixating capacities of autoantibodies in anti-p200 pemphigoid compared with BP suggest disease-specific complement activation signatures and potentially distinct therapeutic thresholds.”

Remark & Suggestion: This sentence contains multiple layered concepts. Consider splitting this sentence into two to clearly distinguish mechanistic insight from therapeutic implications.

Response: We thank the reviewer for the valuable comments regarding language improvement. All relevant sections have been revised accordingly in the updated manuscript.

My General Recommendation: Minor revision

The authors are to be congratulated on a carefully conducted and thoughtfully presented study. Addressing the points above would further enhance clarity and translational impact, but no additional experiments are required.

Response: We thank the reviewer for the constructive comments and positive feedback on our manuscript.

 

Reviewer 3 Report

Comments and Suggestions for Authors

 

Authors wished to investigate the histopathological differences between anti-p200 pemphigoid  and bullous pemphigoid and the  role of complement activation and its pharmacological modulation in vitro . The manuscript is intersting as complement has recently become the target of new therapies.

The following points should be addressed in a revised version of the manuscript.

 

1-Authors should compare the histopathological findings of anti-p200 pemphigoid  to the histopathological findings of  bullous pemphigoid and not simply report the findings of anti-p200 pemphigoid  biopsies.

 

2-In Conclusion Authors state that complement-fixating capacities of autoantibodies is distinct in anti-p200 pemphigoid compared with BP. Actually it seems to me that there is heterogeneity among both anti-p200 and BP, suggesting that complement may play an important role in a subgroup of patients with anti -p200 as well BP.

Author Response

The following points should be addressed in a revised version of the manuscript.

1-Authors should compare the histopathological findings of anti-p200 pemphigoid to the histopathological findings of bullous pemphigoid and not simply report the findings of anti-p200 pemphigoid biopsies.

Response: We thank the reviewer for this constructive comment. As the histopathological features of BP have been well characterized in the literature, several examples of which are cited in the manuscript, the present study primarily focused on the histopathological findings observed in anti-p200 pemphigoid. Nevertheless, relevant comparisons with BP, highlighting key differences in histopathological features, have now been briefly expanded in the Discussion section.

2-In Conclusion Authors state that complement-fixating capacities of autoantibodies is distinct in anti-p200 pemphigoid compared with BP. Actually, it seems to me that there is heterogeneity among both anti-p200 and BP, suggesting that complement may play an important role in a subgroup of patients with anti-p200 as well BP.

Response: We thank the reviewer for this insightful comment. We agree with the reviewer that heterogeneity in complement-fixating capacities of autoantibodies exists in both anti-p200 pemphigoid and BP, although it may be less pronounced in BP disease. Accordingly, specific subgroups in both patient cohorts could potentially benefit from therapies targeting complement activation.

The Discussion section has now been revised accordingly:

“We assessed the complement-fixating potential of anti-p200 pemphigoid IgG using the CFT and examined pharmacological blockade of autoantibody-induced complement activation in vitro. CFT is a well-established assay long applied to the study of BP and pemphigoid gestationis [31]. In this assay, complement-inactivated patient sera were incubated with healthy human skin cryosections. Normal human hirudin plasma was then added as a complement source, with or without specific inhibitors. Among the 50 an-ti-p200 pemphigoid sera, 20 (40%) induced BMZ complement fixation, compared with 13 of 15 (87%) BP sera, indicating a stronger C3-fixating capacity of BP autoantibodies. This difference is consistent with trends observed in DIF studies, in which C3 deposition is re-ported slightly more frequently in BP than in anti-p200 pemphigoid [12, 23, 30]. Both cohorts could be classified as strongly, mildly, or non-complement-fixing subgroups. This heterogeneity, more pronounced in anti-p200 pemphigoid, supports a pathogenic role for complement while highlighting interindividual variability in effector mechanisms. It also suggests that CFT could potentially identify complement-dependent subsets suitable for targeted therapy. Since sera were collected at initial diagnosis during active disease before systemic immunosuppression, the observed variability is unlikely to be attributable to treatment effects or disease chronicity. Whether complement-fixing capacity correlates with disease severity, mucosal involvement, or epitope specificity (e.g., laminin β4 vs. laminin γ1) remains unknown and warrants investigations in future longitudinal studies.”

“While average complement-fixating capacities differ between the two diseases, potentially reflecting distinct therapeutic thresholds, subsets of patients in both cohorts with moderate to strong complement-fixating capacities could benefit from complement-targeted approaches.”