Ana Cardus Figueras

Aims: Microglia undergo profound structural and functional changes during Alzheimer's disease, yet the earliest stages of morphological remodelling that occur prior to amyloid deposition remain poorly defined. We hypothesised that microglia in the hippocampus of App ^NL‐G‐F mice would exhibit early, region‐specific structural adaptations before local plaque formation, reflecting an initial phase of disease‐associated structural remodelling. Methods: Two‐month‐old App ^NL‐G‐F and wildtype mice were examined using high‐resolution confocal microscopy of Iba1‐labelled microglia in the dorsal CA1 apical field. Automated three‐dimensional reconstructions were generated in Imaris, and quantitative morphometric analyses quantified cell density, Iba1 coverage, process topology and Sholl‐based arbor complexity. Statistical analyses were performed using linear mixed‐effects models incorporating sex as a fixed factor in all analyses. Results: Microglial density and total Iba1 coverage were unaffected in App ^NL‐G‐F mice at this age. In contrast, Sholl analysis revealed significant genotype‐dependent reductions in process intersections and total process length, accompanied by reduced individual‐cell territorial coverage, indicating an early contraction of the surveillance arbor independent of cell number. Conclusions: These findings demonstrate that hippocampal microglia in App ^NL‐G‐F mice undergo an early, coordinated structural remodelling before local amyloid deposition becomes apparent. This preplaque adaptation defines an early structural remodelling of hippocampal microglia prior to evident local amyloid deposition, providing new insight into the earliest structural adaptations associated with neuroimmune engagement in AD pathogenesis.

12/15-lipoxygenase (12/15-LOX, Alox15 ) generates bioactive oxygenated lipids during inflammation, however its homeostatic role(s) in normal healing are unclear. Here, the role of 12/15-LOX in resolving skin wounds was elucidated, focusing on how its lipids act together in physiologically relevant amounts. In mice, wounding caused acute appearance of 12/15-LOX-expressing macrophages and stem cells, coupled to early generation of ~12 monohydroxy-oxylipins and enzymatically oxidized phospholipids (eoxPL). Alox15 deletion increased collagen deposition, stem cell/fibroblast proliferation, IL6/pSTAT3, pSMAD3, and interferon (IFN)-γ levels. Conversely, CD206 expression, F480+ cells, and MMP9 and MMP2 activities were reduced. Alox15 ^−/− skin was deficient in PPARγ/adiponectin activity. Furthermore, while pro-inflammatory genes were upregulated as normal during wounding, many including Il6, Il1b, ccl4, Cd14, Cd274, Clec4d, Clec4e, Csf3, Cxcl2, and miR-21 failed to revert to baseline during healing, indicating disruption of PPARγ’s anti-inflammatory brake on NLRP3/inflammasome and TGF-β signaling. Reconstituting Alox15 ^−/− wounds with a physiological mixture of Alox15 -derived primary oxylipins generated by healing wounds restored MMP and dampened collagen deposition. The oxylipin mixture activated the PPARγ response element in vitro, while in vivo, its coactivator, Helz2 , was significantly upregulated as well as several fatty acid and prostaglandin PPARγ ligands. Additional inflammatory and proliferative gene networks impacted by Alox15 ^−/− included Elf4, Cebpb , and Tcf3. In summary, 12/15-LOX generates abundant monohydroxy oxylipins that act together via PPARγ. The identification of multiple gene alterations reveals several targets for treating nonhealing wounds. Our studies demonstrate that 12/15-LOX oxylipins act in concert, dampening inflammation in vivo, revealing the need to consider lipid signaling holistically.

Inhibitory CD4⁺ T cells have been linked with suboptimal immune responses against cancer and pathogen chronicity. However, the mechanisms that underpin the development of these regulatory cells, especially in the context of ongoing antigen exposure, have remained obscure. To address this knowledge gap, we undertook a comprehensive functional, phenotypic, and transcriptomic analysis of interleukin (IL)-10-producing CD4⁺ T cells induced by chronic infection with murine cytomegalovirus (MCMV). We identified these cells as clonally expanded and highly differentiated T_H1-like cells that developed in a T-bet-dependent manner and coexpressed arginase-1 (Arg1), which promotes the catalytic breakdown of L-arginine. Mice lacking Arg1-expressing CD4⁺ T cells exhibited more robust antiviral immunity and were better able to control MCMV. Conditional deletion of T-bet in the CD4⁺ lineage suppressed the development of these inhibitory cells and also enhanced immune control of MCMV. Collectively, these data elucidated the ontogeny of IL-10-producing CD4⁺ T cells and revealed a previously unappreciated mechanism of immune regulation, whereby viral persistence was facilitated by the site-specific delivery of Arg1.

Cytokines that signal via STAT1 and STAT3 transcription factors instruct decisions affecting tissue homeostasis, antimicrobial host defense, and inflammation-induced tissue injury. To understand the coordination of these activities, we applied RNA sequencing, chromatin immunoprecipitation sequencing, and assay for transposase-accessible chromatin with high-throughput sequencing to identify the transcriptional output of STAT1 and STAT3 in peritoneal tissues from mice during acute resolving inflammation and inflammation primed to drive fibrosis. Bioinformatics focused on the transcriptional signature of the immunomodulatory cytokine IL-6 in both settings and examined how profibrotic IFN-γ–secreting CD4+ T cells altered the interpretation of STAT1 and STAT3 cytokine cues. In resolving inflammation, STAT1 and STAT3 cooperated to drive stromal gene expression affecting antimicrobial immunity and tissue homeostasis. The introduction of IFN-γ–secreting CD4+ T cells altered this transcriptional program and channeled STAT1 and STAT3 to a previously latent IFN-γ activation site motif in Alu-like elements. STAT1 and STAT3 binding to this conserved sequence revealed evidence of reciprocal cross-regulation and gene signatures relevant to pathophysiology. Thus, we propose that effector T cells retune the transcriptional output of IL-6 by shaping a regulatory interplay between STAT1 and STAT3 in inflammation.

Cytokines that signal via STAT1 and STAT3 transcription factors instruct decisions affecting tissue homeostasis, anti-microbial host defense, and inflammation-induced tissue injury. To understand the coordination of these activities, we applied RNA-seq, ChIP-seq, and ATAC-seq to identify the transcriptional output of STAT1 and STAT3 in peritoneal tissues during acute resolving inflammation and inflammation primed to drive fibrosis. Bioinformatics focussed on the transcriptional signature of the immuno-modulatory cytokine IL-6 in both settings and examined how pro-fibrotic IFNγ-secreting CD4 ⁺ T-cells altered the interpretation of STAT1 and STAT3 cytokine cues. In resolving inflammation, STAT1 and STAT3 cooperated to drive stromal gene expression affecting anti-microbial immunity and tissue homeostasis. The introduction of IFNγ-secreting CD4 ⁺ T-cells altered this transcriptional program and channeled STAT1 and STAT3 to a previously latent GAS motif in Alu -like elements. STAT1 and STAT3 binding to this conserved sequence revealed evidence of reciprocal cross-regulation and gene signatures relevant to pathophysiology. Thus, we propose that effector T-cells re-tune the transcriptional output of IL-6 by shaping a regulatory interplay between STAT1 and STAT3 in inflammation.

The cytokine IL-6 controls the survival, proliferation and effector characteristics of lymphocytes through activation of the transcription factors STAT1 and STAT3. While STAT3 activity is an ever-present feature of IL-6 signaling in CD4⁺ T cells, prior activation via the T cell antigen receptor limits IL-6’s control of STAT1 in effector and memory populations. Here we found that phosphorylation of STAT1 in response to IL-6 was regulated by the tyrosine phosphatases PTPN2 and PTPN22 expressed in response to the activation of naïve CD4⁺ T cells. Transcriptomics and chromatin immunoprecipitation–sequencing (ChIP-seq) of IL-6 responses in naïve and effector memory CD4⁺ T cells showed how the suppression of STAT1 activation shaped the functional identity and effector characteristics of memory CD4⁺ T cells. Thus, tyrosine phosphatases induced by the activation of naïve T cells determine the way activated or memory CD4⁺ T cells sense and interpret cytokine signals.