Latent Toxoplasma gondii Infection Does Not Modulate Immune Aging in a Cross-Sectional Working-Age Population Study

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

A 19% similarity was detected with the iThenticate program; it is suggested that the manuscript be reviewed.

Line 12.  “For evaluating a potential role of latent T. gondii” Does it refer to a latent T. gondii infection? If so, please specify.

Line 19.  “T. gondii exhibited significant bivariate associations with” ……; Do you mean T. gondii infection? If so, please specify. Same in line 22… and throughout the entire manuscript

Discussion and conclusion sections:

In the study age bias does not invalidate results, but it reduces the weight of the evidence.

Age explains both the higher seroprevalence and the immunological changes. It seems that the results suggest that immune aging reflects a normal, age-related process rather than an effect of latent infection, also it seems that the study can´t determine whether T. gondii infection influences immune aging over time. it would be advisable to reformulate the discussion and conclusions to emphasize that the observed immunosenescence probably reflects an age-related physiological process, rather than an effect of a chronic T. gondii infection.

Adding a short biological explanation of why latent infections may not disrupt immune balance in healthy individuals would help contextualize the findings.

The comparison with acute conditions (sepsis, COVID-19) is interesting but not very relevant to the study's objective, since those contexts involve extreme systemic inflammation. This may distract from the main conclusion: “Latent T. gondii does not modulate immunosenescence in healthy adults”.

Author Response

Reviewer 1:

We would like to thank this reviewer for the insightful hints provided for improving our manuscript. We have addressed all comments in detail as indicated below.

A 19% similarity was detected with the iThenticate program; it is suggested that the manuscript be reviewed.

This study relies on previously established and reported methods, which in our opinion might explain the percentage similarity mentioned above. The changes and additions to the methods requested during this revision (cf. Reviewer 2) will contribute to reducing this amount to a more adequate level.

Line 12.  “For evaluating a potential role of latent T. gondii” Does it refer to a latent T. gondii infection? If so, please specify.

Following this comment, we added “infection” after “T. gondii” where appropriate.

Line 19.  “T. gondii exhibited significant bivariate associations with” ……; Do you mean T. gondii infection? If so, please specify. Same in line 22… and throughout the entire manuscript

Following these comments, we checked the abstract and text and used “latent T. gondii infection” or “T. gondii status” where appropriate.

Discussion and conclusion sections:

In the study age bias does not invalidate results, but it reduces the weight of the evidence.

We agree to this comment, and we have modified the discussion and conclusion section accordingly, also considering similar comments received from Reviewer 2 concerning the weighting of the bivariate associations.

Age explains both the higher seroprevalence and the immunological changes. It seems that the results suggest that immune aging reflects a normal, age-related process rather than an effect of latent infection, also it seems that the study can´t determine whether T. gondii infection influences immune aging over time. It would be advisable to reformulate the discussion and conclusions to emphasize that the observed immunosenescence probably reflects an age-related physiological process, rather than an effect of a chronic T. gondii infection.

We acknowledge this relevant hint and have accordingly included a corresponding statement in the conclusion:

At present, the outcomes of this cross-sectional analysis suggest that the observed immunosenescence probably reflects an age-related physiological process and the latent stage of T. gondii will be unlikely to modulate immune aging in terms of cellular senescence in otherwise healthy working-age adults. If confirmed by prospective longitudinal studies, this trait of the latent form of T. gondii would, if not promote, but at least not compromise survival of host and parasite.

Adding a short biological explanation of why latent infections may not disrupt immune balance in healthy individuals would help contextualize the findings.

In addition to the modified conclusions mentioned above, we added corresponding findings from a recent study to the discussion, stating:

Notably, a recent study suggested that the pressure of immune surveillance on the latent form of T. gondii persisting in long-lived cysts might mitigate infection-induced damage and, thus, promote survival of host and parasite [63].

The comparison with acute conditions (sepsis, COVID-19) is interesting but not very relevant to the study's objective, since those contexts involve extreme systemic inflammation. This may distract from the main conclusion: “Latent T. gondii does not modulate immunosenescence in healthy adults”.

Following this hint, we have reformulated the concerning parts of the manuscript and moved them to a new sub-section “4.1 Outlook”, so that they will no longer distract from the main conclusion.

4.1 Outlook

In contrast to our findings, recent reports on groups affected by acute sepsis [30] and severe SARS-CoV-2-infection [31] showed no age trends for immunosenescence biomarkers, which were also derived from the IMM-AGE metric [22] and, thus, bearing close resemblance to IMMAX [32]. Therefore, it will be relevant to examine whether the correlation of immunosenescence biomarkers with age observed in this study under latent T. gondii infection would also persist under acute or recently acquired T. gondii infection, which had been reported to modulate PBMC subpopulations indicative for cellular senescence, such as NK cells, CD4+ and CD8+ T cells as well as their memory sub-types [40-45].

Reviewer 2 Report

Comments and Suggestions for Authors

The study analyzes 584 adults (20–70 y) from the Dortmund Vital Study, relating T. gondii IgG serostatus to a validated composite immunosenescence index (IMMAX) and its component flow-cytometry markers.Crude comparisons suggest higher immune age among seropositive participants, but all associations vanish after adjusting for age and sex; the T. gondii x age interaction is null, so the age slope of immunosenescence appears unmodified.

Main concerns

in a working-age, generally healthy cohort, latent toxoplasmosis probably does not accelerate cellular immune aging.

potential unmeasured confounding (notably CMV), limited QC disclosure for cytometry, and modest exploration of nonlinear age effects or multiplicity control across many endpoints.

Serostatus is defined by one ELISA IgG with manufacturer cutoffs; one “intermediate” value (33 IU/ml) was assigned “positive”. Please justify this override and show how re-classifying the gray zone as missing/indeterminate changes results (re-run ANCOVA).

Were plates randomized by age/sex/T. gondii? Any duplicate samples or external controls across runs? How many samples near the cutoffs (30–35 IU/ml), and are conclusions robust if a +/-10% window is excluded?

CMV is a canonical driver of T-cell aging (CD8⁺CD28⁻ expansion, naive loss). Without CMV serostatus, the adjusted “null” could be masked confounding if CMV correlates with T. gondii and age. Even a subset analysis (if CMV is available in DVS) or a quantitative bias analysis (plausible CMV prevalences and effect sizes) would be valuable. Authoprs should consider also EBV as sensitivity

Is CMV IgG available for at least a sub-cohort? If not, can you present a bounding analysis to show whether any realistic CMV imbalance could reverse the null?

IMMAX vs age is treated linearly in ANCOVA, but prevalence and many immune markers show nonlinear age patterns. Please re-estimate with splines (3–4 df) and test T. gondii x spline(age); include partial effect plots with 95% CI. For the central claim (“no modification of age trend”), power for an interaction should be reported

Authorts  check heteroscedasticity and influence points across age extremes? Are results stable in age-restricted bands (example, 30–60 y)?

Many endpoints are tested (IMMAX+5 composites+12 percentages). Even if the main claim rests on IMMAX, please present a multiplicity strategy (FDR across secondaries) and avoid over-interpretation of unadjusted bivariate P-values. For adjusted models, emphasize effect sizes with 95% CI (already partly shown) and add standardized mean differences between groups for transparency.a compact forest plot of adjusted contrasts (T.gondii +vs-) across markers with FDR q values.

Were cytometer settings standardized with CS&T beads? Any Levey–Jennings tracking for key populations (% naive CD8, % CD8⁺CD28⁻)? Were analysts blinded to serostatus?  ..

What is the 95% CI around the interaction coefficient for IMMAX, in interpretable units (ΔIMMAX per decade)?.,

The paper narrative connects T. gondii to inflammaging, but no soluble inflammation markers (likr CRP, IL-6, TNFR1) are analyzed. If available in DVS, include at least CRP as secondary outcome or as a covariate; if not, explicitly acknowledge as limitation, are cytokines available at baseline? If yes, do they show any T. gondii association after age/sex adjustment? Please discuss

 

Minor concerns

Consider a DAG showing the causal structure (Age→{T. gondii, IMMAX}; Sex→IMMAX; CMV→IMMAX; T. gondii↔CMV). a methods flowchart (sampling→ ELISA→ PBMC→ IMMAX) also could help-

one-page forest for adjusted effects would reduce text load.

run a probabilistic classification for serostatus near the cutoff (assign 0.25/0.5/0.75 probabilities) and examine whether the adjusted contrast for IMMAX remains near cero.

Keep “immune ageing” vs “immunosenescence” consistent; minor grammar polish is enough (the English I consider  already clear).

The article states data on request; please add a minimal data dictionary (variables, units) and a script snippet for reproducing IMMAX and the main ANCOVA.-

 

Comments on the Quality of English Language

minor grammar polish is enough

Author Response

Please see the attached file for responses.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

none

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have adequately answered the questions posed, and I believe their work contributes to the immunology of T. gondii. Suitable for publication