Fig 2: T cell effector and memory responses of persistent patients(A) Frequencies of CD4+ T lymphocytes producing IFNγ, TNFα and IL-6 and CD8+ T lymphocytes producing IFNγ, TNFα and Granzyme B (Grz B) after polyclonal in vitro stimulation of PBMCs from NI, NP and P (≤10 DSSO) with anti-CD3/CD28 beads of PBMCs from (n = 12), NP (n = 11) and P (n = 23).(B) Longitudinal immunophenotyping of PBMC from NP and P depicting CD4+CD27+CD45RA+CCR7+ naive T cells (naive CD4), CD4+CD27+CD45RA−CCR7- effector memory T cells (EM1) and CD8+CD27+CD45RA + −CCR7+ naive T cells (naive CD8). Filled dots represent individual samples longitudinally collected until resolution of infection from NP and P patients positive by qRT-PCR for SARS-CoV-2. Empty dots represent samples from convalescent patients coinciding with the first negative qRT-PCR for SARS-CoV-2.(C and D) Immunophenotyping of SARS-CoV-2 reactive CD4 and CD8 T cells after in vitro stimulation of PBMC from NP (n = 11) and P (n = 9) after viral clearance with peptides spanning the Spike protein of the alpha variant of SARS-CoV-2 in the presence of IL-2. Percentage of (C) CD4+ and CD8+ cells expressing Ki67, (D) CD4+ cells expressing TNFα, CD8+ cells expressing IFNγ and Granzyme B (Grz B), and CD4+CD25+ cells expressing IL-10. Statistical significance was calculated using Kruskal-Wallis analysis followed by Dunn post-test, and indicated by ∗p ≤ 0.05; ∗∗p ≤ 0.01. DSSO, Days since symptom onset.

Fig 3: Effects of AAA-derived metabolites on human monocytes(A)We treated freshly isolated peripheral blood mononuclear cells from HCs with either vehicle or escalating doses of AAA-derived metabotoxins (in duplicate), identified from our initial metabolomics analyses, for 24 h.(B). We then used multiparametric flow cytometry to evaluate the change in pro-inflammatory cytokine production from monocytes (CD14high) and noted an increase in TNF-α production with increasing doses of IAA; a representative plot is shown here.(C) Quantification of the increase in the proportion of TNF-α-producing monocytes with IAA treatment (n = 9). For all boxplots, the center line indicates the median, the box indicates the 25th and 75th percentiles, the whiskers indicate 1.5× interquartile range (IQR), and the dots indicate outliers.(D) We isolated CD14+ monocytes from HCs and treated them with either vehicle or escalating doses of AAA-derived lactate metabolites (in duplicate), either overnight and then evaluated endocytosis of flurophore-labeled dextran, or for 36 h and then assessed cytokine production.(E) We used flow cytometry to evaluate endocytosis of AF647-dextran by monocytes and noted an increase with indolelactate (ILA) treatment; a representative plot is shown here.(F) Quantification of proportion of monocytes endocytosing AF647-dextran with ILA treatment (n = 5). The data for the various ILA doses is normalized to vehicle for each individual. Bars represent means and error bars represent standard errors of the mean.(G) Boxplot of proportion of monocytes producing IL-6 with vehicle or various doses of ILA.(H) Boxplot of proportion of monocytes producing IL-1β with vehicle or various doses of ILA. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗∗p < 0.001.Also see Figure S6.

Fig 4: ISG15 regulates inflammation from an intracellular location and independently of ISGylation. (A) Culture medium was collected at different times post-infection for ISG15 detection by dot blot. First row: 0, 3, 6, and 9 hpi; second row: 12, 18, 24, and 30 hpi; third row: 36, 42, and 48 hpi; fourth row: 25, 50, 100, and 200 ng/mL of rhISG15. (B) ISG15-KO HeLa cells were pre-treated with the indicated concentration of rhISG15 for 30 min before Chlamydia infection (MOI = 1). The cells were further incubated for 42 h in the presence of rhISG15 before RNA extraction and quantification of IL6 and IL8 expression by RT-qPCR. (C) HeLa cells were incubated with C. trachomatis (MOI = 1) for 42 h or with IFN-α/β (10 ng/mL) for 24 h, lysed, and the amount of ISG15 (free form & conjugates) was analyzed by immunoblot on whole cell lysates. (D) Primary cervical epithelial cells were treated with IFNα (10 ng/mL) for 24 h or with C. trachomatis (MOI = 10) for 42 h followed by ISG15 detection. Top panels of C and D show overexposed images of the upper part of the membranes to visualize ISGylated proteins. (E) ISG15-KO cells (C2 and C4) or complemented cells (with ISG15 WT or ΔGG) were infected or not with C. trachomatis (MOI = 1) for 42 h before measuring the transcription of IL6 and IL8. The panel on the left shows ISG15 expression in the complemented cells. The images displayed are representative of two independent experiments in A and of three independent experiments in D using primary cells from three individuals. All other data correspond to three independent experiments. The p-values of unpaired t test between the indicated groups are shown.

Fig 5: ISG15 limits the inflammatory response in the upper FGT of mice infected with C. trachomatis. ISG15-KO and ISG15-WT mice were infected by introducing C. trachomatis into the uterine horn trans-cervically. The upper FGTs were collected at the indicated times post-infection. The upper FGTs from non-infected mice were collected as negative control. Bacterial loads (A) and inflammatory cytokines (B) in FGT homogenates were determined using quantitative PCR and multi-plex ELISA assays, respectively. Each dot represents one animal. (C) Representative FGTs (left) and hydrosalpinx index for six infected animals (right). The smallest interval of the scale in this image represents 0.05 cm. The p-value of unpaired t test between indicated groups are shown. (D) Graphical summary. Chlamydia infection of epithelial cells is sensed by cGas, which activates the STING/TBK1/IRF3 signaling cascade. Direct release of c-di-AMP by the bacteria may also be involved. IRF3 activates transcription of ISG15. Increase in intracellular free ISG15 restricts bacterial proliferation and the transcription of the host inflammatory cytokines IL6 and IL8, independently of ISGylation.