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NOD-Like Receptor Family Pyrin Domain Containing 3 (NLRP3) inflammasome modulation has emerged as a potential therapeutic approach targeting inflammation amplified by pyroptotic innate immune cell death. In diseases characterized by non-cell autonomous neurodegeneration including amyotrophic lateral sclerosis (ALS), the activation of several inflammasomes has been reported. Since functional redundancy can exist among inflammasome pathways, here we investigate the effects of NLRP3 inhibition on NLRP3, NLR family CARD Domain Containing 4 (NLRC4) and non-canonical pathways to understand whether NLRP3 blockade alone can mitigate pro-inflammatory cytokine release and pyroptotic cell death in contexts where single or multiple inflammasome pathways independent of NLRP3 are activated. In this study we do not limit our insights into inflammasome biology by solely relying on the THP-1 monocytic line under the LPS/nigericin-mediated NLRP3 pathway activation paradigm. We assess therapeutic potential and limitations of NLRP3 inhibition in multi-inflammasome activation contexts utilizing various human cellular systems including cell lines expressing gain of function (GoF) mutations for several inflammasomes, primary human monocytes, macrophages, healthy and Amyotrophic Lateral Sclerosis (ALS) patient induced pluripotent stem cells (iPSC)-derived microglia (iMGL) stimulated for canonical and non-canonical inflammasome pathways. We demonstrate that NLRP3 inhibition can modulate the NLRC4 and non-canonical inflammasome pathways; however, these effects differ between immortalized, human primary innate immune cells, and iMGL. We extend our investigation in more complex systems characterized by activation of multiple inflammasomes such as the SOD1 G93A mouse model. Through deep immune phenotyping by single-cell mass cytometry we demonstrate that acute NLRP3 inhibition does not ameliorate spinal cord inflammation in this model. Taken together, our data suggests that NLRP3 inhibition alone may not be sufficient to address dynamic and complex neuroinflammatory pathobiological mechanisms including dysregulation of multiple inflammasome pathways in neurodegenerative disease such as ALS.

Objectives Pro‐ and anti‐inflammatory properties have been attributed to interleukin‐27 (IL‐27). Nevertheless, the impact of this cytokine on chronic inflammatory diseases such as multiple sclerosis (MS) remains ill‐defined. We investigated the biology of IL‐27 and its specific receptor IL‐27Rα in MS patients. Methods Levels of IL‐27 and its natural antagonist (IL‐27‐Rα) were measured by ELISA in biological fluids. CD4+ and CD8+ T lymphocytes were isolated from untreated relapsing–remitting MS patients and healthy donors. Transcriptome‐wide analysis compared T‐cell subsets stimulated or not with IL‐27. Expression of the IL‐27Rα, key immune factors, STAT phosphorylation and cytokine production was assessed by flow cytometry. Results We observed elevated levels of IL‐27 in the serum and cerebrospinal fluid of MS patients compared with controls. Moreover, we show that specific IL‐27‐mediated effects on T lymphocytes are reduced in MS patients including the induction of PD‐L1. IL‐27‐triggered STAT3 signalling pathway is enhanced in CD4+ and CD8+ T lymphocytes from MS patients. Elevated IL‐27Rα levels in serum from MS patients are sufficient to impair the capacity of IL‐27 to act on immune cells. We demonstrate that shedding of IL‐27Rα by activated CD4+ T lymphocytes from MS patients contributes to the increased IL‐27Rα peripheral levels and consequently can dampen the IL‐27 responsiveness. Conclusion Our work identifies several mechanisms that are altered in the IL‐27/IL‐27R axis in MS patients, especially in T lymphocytes. Our results underline the importance of characterising the biology of cytokines in human patients prior to design new therapeutics. T lymphocytes play a central role in the pathobiology of chronic inflammatory diseases including multiple sclerosis (MS). We identified perturbations in the IL‐27/IL‐27R pathway in MS patients; IL‐27 triggers higher STAT3p response in T lymphocytes from MS vs. controls and induces PD‐L1 less efficiently on T cells from MS patients. Moreover, elevated IL‐27Rα levels in serum from MS patients, shed from activated CD4 T lymphocytes, are sufficient to impair the capacity of IL‐27 to act on immune cells.

Background Interleukin-27 (IL-27) can trigger both pro- and anti-inflammatory responses. This cytokine is elevated in the central nervous system (CNS) of multiple sclerosis (MS) patients, but how it influences neuroinflammatory processes remains unclear. As astrocytes express the receptor for IL-27, we sought to determine how these glial cells respond to this cytokine and whether such exposure alters their interactions with infiltrating activated T lymphocytes. To determine whether inflammation shapes the impact of IL-27, we compared the effects of this cytokine in non-inflamed and inflamed conditions induced by an IL-1β exposure. Main body Transcriptomic analysis of IL-27-exposed human astrocytes showed an upregulation of multiple immune genes. Human astrocytes increased the secretion of chemokines (CXCL9, CXCL10, and CXCL11) and the surface expression of proteins (PD-L1, HLA-E, and ICAM-1) following IL-27 exposure. To assess whether exposure of astrocytes to IL-27 influences the profile of activated T lymphocytes infiltrating the CNS, we used an astrocyte/T lymphocyte co-culture model. Activated human CD4 + or CD8 + T lymphocytes were co-cultured with astrocytes that have been either untreated or pre-exposed to IL‑27 or IL-1β. After 24 h, we analyzed T lymphocytes by flow cytometry for transcription factors and immune molecules. The contact with IL-27-exposed astrocytes increased the percentages of T-bet, Eomes, CD95, IL-18Rα, ICAM-1, and PD-L1 expressing CD4 + and CD8 + T lymphocytes and reduced the proportion of CXCR3-positive CD8 + T lymphocytes. Human CD8 + T lymphocytes co-cultured with human IL-27-treated astrocytes exhibited higher motility than when in contact with untreated astrocytes. These results suggested a preponderance of kinapse-like over synapse-like interactions between CD8 + T lymphocytes and IL-27-treated astrocytes. Finally, CD8 + T lymphocytes from MS patients showed higher motility in contact with IL-27-exposed astrocytes compared to healthy donors’ cells. Conclusion Our results establish that IL-27 alters the immune functions of human astrocytes and shapes the profile and motility of encountered T lymphocytes, especially CD8 + T lymphocytes from MS patients.

CD4 + CD8 + T lymphocytes account for 1–2% of circulating human T lymphocytes, but their frequency is augmented in several diseases. The phenotypic and functional properties of these T lymphocytes are still ill-defined. We performed an ex vivo characterization of CD4 + CD8 + T lymphocytes from the blood of healthy individuals. We observed that CD4 + CD8 + T lymphocytes exhibit several characteristics associated with memory T lymphocytes including the expression of chemokine receptors (e.g. CCR7, CXCR3, CCR6) and activation markers (e.g. CD57, CD95). Moreover, we showed that a greater proportion of CD4 + CD8 + T lymphocytes have an enhanced capacity to produce cytokines (IFNγ, TNFα, IL-2, IL-4, IL-17A) and lytic enzymes (perforin, granzyme B) compared to CD4 + and/or CD8 + T lymphocytes. Finally, we assessed the impact of three key cytokines in T cell biology on these cells. We observed that IL-2, IL-7 and IL-15 triggered STAT5 phosphorylation in a greater proportion of CD4 + CD8 + T lymphocytes compared to CD4 and CD8 counterparts. We demonstrate that CD4 + CD8 + T lymphocytes from healthy donors exhibit a phenotypic profile associated with memory T lymphocytes, an increased capacity to produce cytokines and lytic enzymes, and a higher proportion of cells responding to key cytokines implicated in T cell survival, homeostasis and activation.