Interleukin‐12/23 deficiency differentially affects pathology in male and female Alzheimer's disease‐like mice

Abstract Pathological aggregation of amyloid‐β (Aβ) is a main hallmark of Alzheimer's disease (AD). Recent genetic association studies have linked innate immune system actions to AD development, and current evidence suggests profound gender differences in AD pathogenesis. Here, we characterise gender‐specific pathologies in the APP23 AD‐like mouse model and find that female mice show stronger amyloidosis and astrogliosis compared with male mice. We tested the gender‐specific effect of lack of IL12p40, the shared subunit of interleukin (IL)‐12 and IL‐23, that we previously reported to ameliorate pathology in APPPS1 mice. IL12p40 deficiency gender specifically reduces Aβ plaque burden in male APP23 mice, while in female mice, a significant reduction in soluble Aβ1–40 without changes in Aβ plaque burden is seen. Similarly, plasma and brain cytokine levels are altered differently in female versus male APP23 mice lacking IL12p40, while glial properties are unchanged. These data corroborate the therapeutic potential of targeting IL‐12/IL‐23 signalling in AD, but also highlight the importance of gender considerations when studying the role of the immune system and AD.

Thank you for the submission of your manuscript to EMBO reports. We have now received the enclosed referee reports on it.
As you will see, while the referees acknowledge that the gender-specific effects are interesting, both referees 2 and 3 point out that in the absence of a little more mechanistic insight the paper will not be a good fit for EMBO reports. Both referees suggest that microglia activation around the plaques should be analyzed, in addition to a few other suggestions.
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Referee #1:
Eede, Obst, et al report important gender differences in the APP23 mice, a slow model of AD. Specifically, female mice show more amyloid formation; moreover, the authors also investigate how gender differences impact the effects of IL-12p40 deficiency on amyloidosis. They show that in male mice, IL-12p40 deficiency reduces plaque burden, whereas in female mice it reduced soluble Aβ species. This study extends previous work on the impact of gender in amyloidosis and shows an important gender difference in the impact of IL-12/IL-23 signaling in the response to amyloidosis. I find the paper highly significant, as it highlights the importance of gender considerations when studying AD and, particularly, the role of immune responses to AD. Moreover, as opposed to previous papers, this paper studies gender differences in a slow AD model (APP23), which may be more physiological than previously examined fast models of AD. Thus, the paper provides significant advances to the field. It is extremely carefully performed, very detailed and well written. I think it will be extremely impactful in the AD field and neurodegeneration, in general. I have only one minor point that the authors may consider. If mouse tissue is still available, the authors may consider evaluating neurite dystrophy by staining for NT-APP or LAMP1. This analysis would directly indicate the impact of the amyloid on neuronal functions.

Referee #2:
The authors expand on previous published data showing that deletion of IL12p40 mitigates amyloid plaque deposition, demonstrating this in a different mouse model (APP23, slower plaque formation than the previous APPPS1), and demonstrating an interesting effect of sex. Namely, they find that IL12p40 deletion has less impact on plaques in female APP23 mice, which have more plaque deposition and in particular more fibrillar plaques. The paper has several strengths. The characterization of male vs. female APP23 at advanced ages is very thorough in terms of Abeta species. The effect of IL12p40 deletion is impressive, and this represents a viable therapeutic target, and is thus valuable. Finally, the sex difference in the effect of IL12p40 is interesting and has important implications. However, there are some concerns: 1. The paper is not particularly novel. The effects of sex on plaques are well known, as are the effects of IL12p40 deletion. While this paper extends these findings to a new model and examines sex, these are modest advances. Addressing criticism 2 may alleviate this. 2. There is very little mechanistic insight in the paper. Though the authors invoke a possible effect on microglia, there is no analysis of the effects of IL12p40 deletion on glial activation/interaction. There is analysis of some cytokines in tissue, of which only CXCL1 is different, but the paper would be much stronger if some morphologic analysis of microglial activation around plaques with Iba1/Cd68/other markers were performed, as well as staining for astrocyte activation. It remains unclear if this is a differential effect of IL12/23 on female vs. male microglia, or if the effects observed are simply due to the different plaque makeup in females (more fibrillar Abeta). The authors discuss this a bit, but analysis of the degree of peri-plaque microglial activation/lysosome expression in female vs. male IL12p40 KO mice could help determine if there is a differential response to plaques. In vitro Abeta uptake assays in male vs. female microglia stimulated with IL12/23 would also be illustrative, but perhaps beyond the scope of a Report. In general, this is a technically strong paper, but the novelty of the observation does not make up for the lack of mechanistic insight. Thus, in my opinion, a bit more detailed analysis of glial responses is needed.

Referee #3:
Gender-specific pathology is differentially affected by interleukin-12/23 subunit deficiency in male and female Alzheimer's disease-like mice Pascale Eede1,{section sign}, Juliane Obst1,2,{section sign}, Annett Böddrich3, Erich E. Wanker3, Stefan Prokop1,4,{section sign}, Frank L. Heppner1,5,6,7,{section sign},* This paper follows on from vom Berg et al in 2012 by examining the impact of IL-12p40 deletion in a model of Alzheimer's disease. While the first paper looked at APP/PS1 mice, this current article examines the impact in APP23 mice, a slower progressing animal model of disease. The authors demonstrate that deletion of IL-12p40 is protective in male APP23 mice only, reducing plaque burden in this group alone. This article is well written, and the results on gender differences are very timely however, this reviewer thinks that more experiments are needed to determine the reason behind the sex-dependent difference in their findings and more mechanistic data would improve the impact of this finding.
1) The mice used are 21 months old, this is a very late stage for mice, and it would be interesting to know if the mortality is the same in male and females. Are we already seeing a survivor effect? 2) In the vom Berg paper, it states that equal numbers of male and female mice are used, yet no gender differences were observed in the APP/PS1 mice after IL-12p40 deletion (even though it has been reported that there are gender differences in plaque load in APP/PS1 mice). It would be interesting to hear the authors take on this finding in contrast with the APP23 model and the findings in this current paper.
3) It would be nice to clearly demonstrate that in APP23 mice, the IL-12p40 is coming from microglia, the authors refer to finding from a different mouse model (APP/PS1 mice) on this point. 4) It would be great to see staining of the microglia around the amyloid plaques in the male vs females. Are the microglia taking up more Abeta? Is the deposition reduced in the ko's? What are the levels of BACE? What is the effect on Abeta degradation enzymes e.g. IDE or Neprilysin? This information would help to understand the sex differences observed in the present study 5) Can the authors rule out a peripheral effect of IL-12p40 deletion? Are there any changes to the peripheral inflammation? The microbiome? , and co-labelled with the 4G8 antibody to stain Ab. Since differences in plaque sizes are a potential confounding element when assessing the amount of plaque-associated BACE1-immunoreactive neurites, we calculated the ratio of the area covered by BACE1 to the area covered by 4G8-positive Ab. The respective results are now included in the revised manuscript in Fig. 2A, 5A & 7A. In summary, these additional data reveal no differences in plaque-associated BACE1 immunoreactivity between male and female APP23 mice harbouring or lacking IL12p40. Reply: We agree with the reviewer's observation that analysing both astrocyte and microglia characteristics could help in understanding our findings relating to plaque pathology in the herein used AD-like mouse models. When quantifying GFAP-positive cortical astrocytes (cells/mm³) we saw increased astrocyte numbers in female APP23 mice compared to male mice, which directly correlated to the increased 4G8-and Congo Red-positive plaque load in female mice ( Fig. 2B; Fig. EV1A in revised manuscript). Upon IL12p40 deletion, we could not detect any differences in astrocyte number in either gender (Fig. 5B & 7B in revised manuscript).
In order to assess microglial characteristics, we quantified plaque-associated microglia using the microglia marker Iba1 and their expression of the activation marker Clec7a (Fig. 2C, 5C & 7C in revised manuscript). This analysis revealed no gender-specific differences or an effect of IL12p40 deletion upon the number of plaque-associated microglia or the presence of Clec7a-positive activated microglia. Instead of studying microglial Aβ uptake in vitro, as suggested by this referee, we performed radial intensity profiling on confocal images of brain tissue to measure 4G8-positive signal within Iba1-positive microglia as an indicator of microglial Aβ uptake. This analysis allowed depicting 4G8-positive amyloid intensity peaks inside the cell (~4 µm) illustrating Aβ uptake by microglia, yet there were no differences in intracellular Aβ levels between male and female APP23 mice harbouring or lacking IL12p40 (Fig. 2D, 5D & 7C in revised manuscript).

Reply:
In order to answer the reviewer's question, we have plotted the age of death (natural or by use in experiment) with respect to gender (Fig. R1). It becomes apparent from this qualitative analysis that male APP23 mice do not tend to die earlier than female mice and are similarly available for the use in experiments requiring aged animals. In our study looking at APP23 and APP23p40 -/mice, we also did not have a drop-out of mice from the experimental groups assigned at early ages of the mice. 2) "In the vom Berg paper, it states that equal numbers of male and female mice are used, yet no gender differences were observed in the APP/PS1 mice after IL-12p40 deletion (even though it has been reported that there are gender differences in plaque load in APP/PS1 mice). It would be interesting to hear the authors take on this finding in contrast with the APP23 model and the findings in this current paper."

Reply:
We agree with the reviewer that gender differences not only appear in the APP23 mouse strain but also in APPPS1 mouse strains. The This confirms that our previously published data on the impact of IL12p40 deletion in APPPS1 mice were not influenced by differences between genders and supports the notion that no differences in cortical Aβ plaque load exist at 4 months in APPPS1-21 mice. It is important to note, however, that several currently used APPPS1 mouse strains differ (i) in the Presenilin mutation used for generating the respective transgenic mouse strain, as well as (ii) in the promotor used for overexpression of transgenes. APPPS1-21 mice used in our previous studies harbour the Thy1 promotor, while transgene expression in other, commonly used APPPS1 models is driven by the Prnp promotor, which may not only account for differences in the quantity and region of transgene expression, but may also have distinct gender-specific effects. Along this line, it is interesting to learn that several previous publications demonstrate substantial gender differences in APPPS1 mouse strains using alternative PS1 mutations and promotor constructs. Taken together, we assume that the gender-specific effect in APP23 mice described by usat least at the investigated age of 21 months -is likely a result of differences in the composition of Aβ amyloid including Aβ plaques as well as in disease progression between males and females.

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3) "It would be nice to clearly demonstrate that in APP23 mice, the IL-12p40 is coming from microglia, the authors refer to finding from a different mouse model (APP/PS1 mice) on this point." Reply: This is a most valid aspect and we have now added a more thorough analysis of IL12p40 expression (Il12b gene) in APP23 mice (Fig. 3A in revised manuscript). Specifically, we isolated RNA (i) from whole brain, (ii) from microglia sorted using CD11bpositive microbeads as well as (iii) the CD11b-negative cell fraction from both male and female APP23 mice. qPCR analysis for the Il12b gene revealed that only the microgliapositive cell fraction showed Il12b expression. No Il12b expression was detected in whole brain and CD11b-negative cell fractions. Microglial Il12b expression also did not differ between male and female APP23 mice, which also correlates with IL12p40 protein expression as measured by ELISA (see Fig. 3B in revised manuscript). Reply: With respect to visualising microglia around Aβ plaques in male and female APP23 mice, we have now added a quantification of plaque-associated microglia using the myeloid cell marker Iba1 as well as an analysis of the microglial activation marker Clec7a (now shown in Fig. 2C, 5C & 7C in revised manuscript). This analysis revealed no gender-specific differences or an effect of IL12p40 deletion upon the number of activated plaque-associated microglia.

4) "It would be great to see staining of the microglia around the amyloid plaques in
We also agree with this referee that measuring microglial Aβ uptake is also of interest and refer to our reply to referee #2 (see above): in brief, radial intensity profiling on confocal images of brain tissue measuring 4G8-positive signals within Iba1-positive microglia as an indicator of microglial Aβ uptake did not reveal differences in intracellular Aβ levels between male and female APP23 mice harbouring or lacking IL12p40 (Fig. 2D, 5D & 7C in revised manuscript). We also performed Western Blot analysis of BACE1, IDE and Neprilysin comparing the effect of IL12p40 deletion in male and female APP23 mice. The results can be found in Fig.  EV2B & EV3B of the revised manuscript. Expression levels of all three proteins were unchanged in APP23 versus APP23p40 -/animals indicating that the sex-specific effect of IL12p40 deletion upon Aβ pathology appears not to be regulated by differences in Aβ processing or degradation. Given that an analysis of the microbiome -despite being fascinating -was not feasible due to the lack of collecting the respective faecal samples during the study, we rather investigated potential IL12p40-mediated effects on peripheral inflammation by analysing plasma samples collected from mice of our study using the 10-plex Pro-inflammatory Panel 1 (mouse) Mesoscale Kit (Fig. EV2A-I, EV2C-K & EV3C-K in revised manuscript). Adding to the differences seen in brain cytokine levels between male and female APP23 mice harbouring or lacking IL12p40, we found that female APP23 mice show significantly increased IL-10 plasma levels compared to male mice. Upon IL12p40 deficiency, peripheral IFNγ levels were found to be decreased in male mice, whilst female APP23p40 -/mice showed both an upregulation of IL1β and CXCL1 as well as a downregulation of IL-5 and IL-6 in their plasma. Such differences in the cytokine milieu in brain and plasma of male and female APP23 may account for the observed gender-specific variation in AD pathogenesis. Interestingly, we also found a correlation between CXCL1 levels in the brain and both soluble and insoluble Aβ 1-40 (Fig. EV1B in revised manuscript). In light of the higher numbers of cortical astrocytes in female APP23 mice, it is interesting to note that astrocytespecific Thank you for the submission of your revised manuscript. We have now received the comments from both referees, and both support its publication now. We can therefore in principle accept your study.
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I would like to suggest some minor changes to the abstract that needs to be written in present tense: Pathological aggregation of amyloid-β (Aβ) is a main hallmark of Alzheimer's disease (AD). Recent genetic association studies have linked innate immune system actions to AD development and current evidence suggests profound gender differences in AD pathogenesis. Here, we characterize gender-specific pathologies in the APP23 AD-like mouse model and find that female mice show stronger amyloidosis and astrogliosis compared with male mice. We tested the gender-specific effect of lack of IL12p40, the shared subunit of interleukin (IL)-12 and IL-23 that we previously reported to ameliorate pathology in APPPS1 mice. IL12p40 deficiency gender-specifically reduces Aβ plaque burden in male APP23 mice, while in female mice a significant reduction of soluble Aβ1-40 without changes in Aβ plaque burden is seen. Similarly, plasma and brain cytokine levels are altered differently in female versus male APP23 mice lacking IL12p40, while glial properties are unchanged. These data corroborate the therapeutic potential of targeting IL-12/IL-23 signalling in common tests, such as t-test (please specify whether paired vs. unpaired), simple χ2 tests, Wilcoxon and Mann-Whitney tests, can be unambiguously identified by name only, but more complex techniques should be described in the methods section; are tests one-sided or two-sided? are there adjustments for multiple comparisons? exact statistical test results, e.g., P values = x but not P values < x; definition of 'center values' as median or average; definition of error bars as s.d. or s.e.m.
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Based on experience of amyloid beta and cytokine measurement of APP23 mice and their crossings, 7-10 animals/genotype were analyzed.
All samples with the right genotype were included. One animal was identified as outlier by Grubbs test and excluded from all analyses. Sample size for brain samples for histology and biochemical analyses as well as serum samples differed due to sample availability. For cytokine analysis in serum some samples were determined as outliers by Grubb's test and excluded. Since the animals used in this study were not treated, no randomization was applied. The only difference between the groups was a genetic alteration.
For histological analyses, the respective investigator was blinded for the gender and genotype of the animals.
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