Fig 2: Transient increase in neuronally-derived estradiol in cultured female cortical neurons. A, left, Western blotting for TSP2 [top, ~160-kDa observed band (~130 kDa expected); bottom, total protein blot for normalization] from astrocyte media collected on DIV10. Right, No difference was found in secreted TSP2 levels between male and female astrocytes; p = 0.568 [unpaired Student’s t test; n = 4 independent experimental replicates (M1-4, F1-4)]. B, left, Western blotting for a2d-1 (top, 143-kDa observed band; bottom, total protein blot for normalization) from purified male and female cortical neuron lysates. Lysates were collected on DIV13 following treatment with NGM only, 100 nm E2, or 100 nm letrozole (Let) on DIV7 and DIV10. Right, No difference in total a2d-1 protein expression was observed between male or female-derived neurons at baseline or following treatment with either E2 or Let; p = 0.805 (two-way ANOVA, interaction: F(2,12) = 0.220; n = 3 independent experimental replicates). C, Immunoassay results for levels of E2 detected in male versus female neuron-conditioned media. An increase in secreted E2 was detected in female cultures at the start of the second week, reaching significance at DIV9; *p < 0.05 [linear mixed-effects model (REML) with Holm–Sidak’s post hoc analysis, sex: F(1,55) = 7.00; n = 4 independent experimental replicates]. D, Estradiol measurements from male versus female cortical ACM. No significant differences were observed (linear mixed-effects model, sex: F(1,29) = 3.80; n = 3 independent experimental replicates).

Fig 3: Increased synaptic response to astrocytic factor TSP2 by male but not female-derived cortical neurons. A, Representative ICC images of male and female-derived cortical neurons. Co-localized presynaptic (Bassoon; magenta) and postsynaptic (Homer1; green) puncta reveal sites of excitatory synapses (white arrowheads). B, No baseline difference in the number of co-localized excitatory synaptic puncta between male and female-derived neurons with standard NGM treatment. The mean difference between M and F is shown in the Gardner–Altman estimation plot. Both groups are plotted on the left axes; the mean difference is plotted on a floating axis on the right as a bootstrap sampling distribution. The mean difference is depicted as a dot; the 95% CI is indicated by the ends of the vertical error bar. C, Sample mEPSC traces from male and female-derived cortical neurons treated with either standard NGM or NGM with the addition of purified TSP2. D, E, TSP2 treatment increased frequency (D) but not amplitude (E) of mEPSCs recorded from male-derived cortical neurons. Female-derived cortical neurons showed no change in mEPSC frequency but had a small decrease in amplitude after TSP2.

Fig 4: Inhibition of neuronal estrogen production modulates TSP/astrocyte-induced synaptogenesis. A, ICC image of a male-derived cortical neuron fixed and stained on DIV13 with presynaptic (Bassoon; magenta) and postsynaptic (Homer1; green) markers. Dotted box indicates example dendritic ROI sampled for images in B, which shows co-localized excitatory synaptic puncta (white, arrowheads) along male and female-derived cortical neurites treated with NGM only or NGM plus TSP2, 100 nm letrozole (Let), or TSP2/letrozole on DIV7 and DIV10. C, left, Letrozole had a mild synaptogenic effect on male-derived cortical neurons but attenuated the synapse-promoting ability of TSP2. Right, Female-derived cortical neurons showed no synaptogenic response to TSP2 unless treatment was combined with Let. D, Delayed treatment (DIV13 and DIV16, fixed and stained DIV19) revealed no differences in synapse number from NGM-only condition following TSP2 and/or Let treatment in neurons from either sex. E, Quantification of Bassoon/Homer1 co-localized synaptic puncta following DIV7/DIV10 treatment schedule showed that Let abolished the synaptogenic effects of sex-matched astrocyte inserts (Astro) and ACM in male-derived neurons as well as ACM in female-derived neurons. No attenuation was observed with Let and Astro in females; *p < 0.05, ***p < 0.001, ****p < 0.0001 [Kruskal–Wallis test with Dunn’s multiple comparisons post hoc analysis; K-W statistic = 52.57 (male), 66.18 (female); n = 30 cells per condition per experimental replicate, 3 independent experimental replicates].

Fig 5: Biomarkers of early fibrotic remodelling, collagen synthesis, and inflammation. (a) Plasma levels of CD163 and the matricellular markers GDF15, MMP1, TSP2, and IGFBP7 were measured in patients with COVID-19, influenza A, and with interstitial lung diseases (UIP, NSIP, and AFE). Mean levels ± standard errors for all time points. Asterisks indicate statistical significance as indicated by bars (*p < 0·05, **p < 0·01, ***p < 0·001). (b) Differential regulation of mRNA expression in lung autopsy tissue of COVID-19 (n = 15) and influenza A (n = 7), and in lung explants from patients with interstitial lung diseases (UIP, n = 6; NSIP, n = 6; AFE, n = 6). Boxplots showing normalized log2 counts of mRNA expression ± standard errors of the mean. Asterisks indicate statistical significance as indicated by bars (*p < 0·05, **p < 0·01, ***p < 0·001).