Preprint Club
A cross-institutional Journal Club Initiative

Keywords

• Sex differences

• T cell differentiation

• Glutaminolysis

Main Findings

The prevalence and severity of immune disease differ between men and women. Especially central to immune responses in many diseases are CD4+ T-cells. Upon activation, they differentiate into Th1, Th2, or Th17 subsets via specific metabolic pathways, with glutaminolysis being key for Th17 and Th2 cell differentiation and function. In this pre-print, Chowdhury et al. found lower glutaminolysis and mitochondrial respiration markers in male Th17 cells from mediastinal lymph node samples obtained from deceased humans. Androgens, which are present at higher levels in males, might affect the expression of these metabolic markers. To explore the potential role of androgens in driving the differences observed in Th17 cells, the authors used a Cre mouse model with CD4-specific androgen receptor deletion in which they induce allergic airway inflammation using house mite dust mite. The authors observed an increase in Th17 cells and metabolic markers like pS6 in male mice compared to the wild type, a response absent in females. In Th17 cells differentiated from splenic CD4+ naïve T cells from male mice expressing a mutated and inactive androgen receptor, mitochondrial respiration was higher when compared to wild-type controls. The authors performed a targeted metabolomic analysis to find the pathway responsible for these differences. In Th17 cells with the mutated receptor, glutamine metabolic pathways were significantly increased. These results were confirmed by Seahorse Substrate Oxidation Assay using the glutaminase inhibitor CB839. In vivo, androgen receptor mutation lessened airway inflammation in females, phenocopying the males. Further, glutamine tracing experiments showed increased uptake in male mice with the mutated receptor. An additional CRISPR screening using Th17 cells differentiated from OT-II Cas9 from male and female mice revealed the glutamine transporter, Asct2, as vital for female Th17 cell fitness. To explore the T Cell differences in severe asthma in humans, the authors used SCENITH, a single-cell metabolomic approach, and demonstrated that male CD4+ T cells take up more glutamine than female CD4+ T Cells, corroborating the mouse findings.

Limitations & Suggestions

• The authors provide two supplementary tables describing the age, race, and ethnicity of the human donors in the study (Tables S1 and S4). We noticed that the race does not match between tables, and ethnicity is not mentioned in Table S4.

  • Causes of death could significantly impact the immune system and it would be interesting to include this information

  • Similarly, reporting the last known medication of the patients would be valuable for the same reasons

• In Figure 1, the authors present a well-executed UMAP visualization of cell surface markers in all samples obtained with mass cytometry. This provides valuable insights and we believe the pre-print could benefit from a more detailed description of this data. For instance, it would be interesting to see how men and women distribute within the plot or a comment about why the expression of markers that normally cluster together (such as CD3 or CD4) results in different clusters.

• In Figure 1, the color scheme that represents mean intensities from the mass cytometry is confusing. We suggest the authors use the more commonly used scheme of white (no expression) to increase color (yellow fully saturated = highest expression).

• In Figure 2, the authors collect bronchoalveolar lavage (BAL) fluid from their mouse models, prepare cytospins, and take light microscopy pictures to count neutrophils and eosinophils among other cells. Total cell numbers are given, but no percentages. Cytospins might be difficult to keep consistent or analyze if the preparation is too full. The authors also mention that the BAL fluid from one mouse could not be collected, supporting our concerns. We wonder if the amount of fluid was the same for all mice, and how consistently could be assessed after the cytospins preparations. It would have been more accurate and reliable to perform flow cytometry on these samples to obtain a better overview of immune cell infiltration.

• In Figure 2C, it was not clear how the data was obtained. Is this before immune activation?

• In Figure 2E-F, only data representative of 2 replicates is given. We suggest the authors include at least 1 additional replicate if possible. Furthermore, additional replicates would be beneficial to other sections of the pre-print, as differences could reach statistical significance (Figure 3).

• In Figure 3, the authors show an increase in mitochondrial respiration in differentiated Th17 cells from mice with a mutated androgen receptor. However, using MitoSox Red measured via flow cytometry, the authors show a reduced ROS production. We find the fact puzzling that increased mitochondrial respiration does not lead to more ROS. The authors could comment on the discrepancy of these observations. During the introduction, the authors comment on the role of glutaminolysis in the production of glutathione, a ROS scavenger. It would be interesting to know the glutathione levels in these samples if they were measured.

• In Figure 3F, the authors show representative transmission electron microscopy pictures to demonstrate ultrastructural differences in mitochondria. We suggest the authors provide quantification of these differences.

• In general, it would be interesting to know if female mice receiving certain doses of testosterone, would they phenocopy the male mice upon immune activation?

We suggest additional analysis:

• Direct comparison of the AR knocked-out male mice to the female mice

• Direct comparison of the GLS knocked-out male mice to the female mice

Significance/Novelty

The pre-print shows the involvement of androgen signaling driving sex differences in Th17 cell activation. The authors provided a mechanistic explanation for this observation: the glutamine metabolism. These results open new exciting questions in the field. How exactly does the androgen signaling cascade regulate metabolic differences in males? Is it merely due to the expression of glutamine transporters? Does androgen signaling control the expression of these genes directly or are the other pathways involved? What other pathways are involved? Women do express androgens and their receptors. What biological threshold exists to explain the observations described in the preprint and how do they work?

This pre-print provides yet another example that biological sex should be considered in immunological studies and encourages immunologist to explore metabolic differences between sexes in their favorite immune cells.

Credit

Reviewed by Sergio San Miguel (Max Planck Institute for Infection Biology) and Dominik Tüchler (Medical University Vienna) as part of a cross-institutional journal club between the Vanderbilt University Medical Center (VUMC), the Max-Delbrück Center Berlin, the Ragon Institute Boston (Mass General, MIT, Harvard), the Medical University of Vienna and other life science institutes in Vienna.

The author declares no conflict of interests in relation to their involvement in the review.