GSDMD is associated with survival in human breast cancer but does not impact anti-tumor immunity in a mouse breast cancer model.

Inflammation plays a pivotal role in cancer development, with chronic inflammation promoting tumor progression and treatment resistance, whereas acute inflammatory responses contribute to protective anti-tumor immunity. Gasdermin D (GSDMD) mediates the release of pro-inflammatory cytokines such as IL-1ß. While the release of IL-1ß is directly linked to the progression of several types of cancers, the role of GSDMD in cancer is less clear. In this study, we show that GSDMD expression is upregulated in human breast, kidney, liver, and prostate cancer. Higher GSDMD expression correlated with increased survival in primary breast invasive carcinoma (BRCA), but not in liver hepatocellular carcinoma (LIHC). In BRCA, but not in LIHC, high GSDMD expression correlated with a myeloid cell signature associated with improved prognosis. To further investigate the role of GSDMD in anticancer immunity, we induced breast cancer and hepatoma tumors in GSDMD-deficient mice. Contrary to our expectations, GSDMD deficiency had no effect on tumor growth, immune cell infiltration, or cytokine expression in the tumor microenvironment, except for Cxcl10 upregulation in hepatoma tumors. In vitro and in vivo innate immune activation with TLR ligands, that prime inflammatory responses, revealed no significant difference between GSDMD-deficient and wild-type mice. These results suggest that the impact of GSDMD on anticancer immunity is dependent on the tumor type. They underscore the complex role of inflammatory pathways in cancer, emphasizing the need for further exploration into the multifaceted effects of GSDMD in various tumor microenvironments. As several pharmacological modulators of GSDMD are available, this may lead to novel strategies for combination therapy in cancer.

Interleukin-38 overexpression in keratinocytes limits desquamation but does not affect the global severity of imiquimod-induced skin inflammation in mice.

Psoriasis is a common chronic inflammatory skin disease that significantly impacts the patients' quality of life. Recent studies highlighted the function of the interleukin (IL)-1 family member IL-38 in skin homeostasis and suggested an anti-inflammatory role for this cytokine in psoriasis. In this study, we generated mice specifically overexpressing the IL-38 protein in epidermal keratinocytes. We confirmed IL-38 overexpression in the skin by Western blotting. We further detected the protein by ELISA in the plasma, as well as in conditioned media of skin explants isolated from IL-38 overexpressing mice, indicating that IL-38 produced in the epidermis is released from keratinocytes and can be found in the circulation. Unexpectedly, epidermal IL-38 overexpression did not impact the global severity of imiquimod (IMQ)-induced skin inflammation, Similarly, keratinocyte activation and differentiation in IMQ-treated skin were not affected by increased IL-38 expression and there was no global effect on local or systemic inflammatory responses. Nevertheless, we observed a selective inhibition of CXCL1 and IL-6 production in response to IMQ in IL-38 overexpressing skin, as well as reduced Ly6g mRNA levels, suggesting decreased neutrophil infiltration. Epidermal IL-38 overexpression also selectively affected the desquamation process during IMQ-induced psoriasis, as illustrated by reduced plaque formation. Taken together, our results validate the generation of a new mouse line allowing for tissue-specific IL-38 overexpression. Interestingly, epidermal IL-38 overexpression selectively affected specific disease-associated readouts during IMQ-induced psoriasis, suggesting a more complex role of IL-38 in the inflamed skin than previously recognized. In particular, our data highlight a potential involvement of IL-38 in the regulation of skin desquamation.

Role of the soluble epoxide hydrolase in keratinocyte proliferation and sensitivity of skin to inflammatory stimuli.

The lipid content of skin plays a determinant role in its barrier function with a particularly important role attributed to linoleic acid and its derivatives. Here we explored the consequences of interfering with the soluble epoxide hydrolase (sEH) on skin homeostasis. sEH; which converts fatty acid epoxides generated by cytochrome P450 enzymes to their corresponding diols, was largely restricted to the epidermis which was enriched in sEH-generated diols. Global deletion of the sEH increased levels of epoxides, including the linoleic acid-derived epoxide; 12,13-epoxyoctadecenoic acid (12,13-EpOME), and increased basal keratinocyte proliferation. sEH deletion (sEH-/- mice) resulted in thicker differentiated spinous and corneocyte layers compared to wild-type mice, a hyperkeratosis phenotype that was reproduced in wild-type mice treated with a sEH inhibitor. sEH deletion made the skin sensitive to inflammation and sEH-/- mice developed thicker imiquimod-induced psoriasis plaques than the control group and were more prone to inflammation triggered by mechanical stress with pronounced infiltration and activation of neutrophils as well as vascular leak and increased 12,13-EpOME and leukotriene (LT) B4 levels. Topical treatment of LTB4 antagonist after stripping successfully inhibited inflammation and neutrophil infiltration both in wild type and sEH-/- skin. While 12,13-EpoME had no effect on the trans-endothelial migration of neutrophils, like LTB4, it effectively induced neutrophil adhesion and activation. These observations indicate that while the increased accumulation of neutrophils in sEH-deficient skin could be attributed to the increase in LTB4 levels, both 12,13-EpOME and LTB4 contribute to neutrophil activation. Our observations identify a protective role of the sEH in the skin and should be taken into account when designing future clinical trials with sEH inhibitors.

Prostaglandin E2 Boosts the Hyaluronan-Mediated Increase in Inflammatory Response to Lipopolysaccharide by Enhancing Lyve1 Expression.

Macrophages are a highly versatile and heterogenic group of immune cells, known for their involvement in inflammatory reactions. However, our knowledge about distinct subpopulations of macrophages and their specific contribution to the resolution of inflammation remains incomplete. We have previously shown, in an in vivo peritonitis model, that inhibition of the synthesis of the pro-inflammatory lipid mediator prostaglandin E2 (PGE2) attenuates efficient resolution of inflammation. PGE2 levels during later stages of the inflammatory process further correlate with expression of the hyaluronan (HA) receptor Lyve1 in peritoneal macrophages. In the present study, we therefore aimed to understand if PGE2 might contribute to the regulation of Lyve1 and how this might impact inflammatory responses. In line with our in vivo findings, PGE2 synergized with dexamethasone to enhance Lyve1 expression in bone marrow-derived macrophages, while expression of the predominant hyaluronan receptor CD44 remained unaltered. PGE2-mediated Lyve1 upregulation was strictly dependent on PGE2 receptor EP2 signaling. While PGE2/dexamethasone-treated macrophages, despite their enhanced Lyve1 expression, did not show inflammatory responses upon stimulation with low (LMW) or high-molecular-weight hyaluronan (HMW)-HA, they were sensitized towards LMW-HA-dependent augmentation of lipopolysaccharide (LPS)-induced inflammatory responses. Thus, Lyve1-expressing macrophages emerged as a subpopulation of macrophages integrating inflammatory stimuli with extracellular matrix-derived signals.

Cell Intrinsic IL-38 Affects B Cell Differentiation and Antibody Production.

IL-38 is an IL-1 family receptor antagonist with an emerging role in chronic inflammatory diseases. IL-38 expression has been mainly observed not only in epithelia, but also in cells of the immune system, including macrophages and B cells. Given the association of both IL-38 and B cells with chronic inflammation, we explored if IL-38 affects B cell biology. IL-38-deficient mice showed higher amounts of plasma cells (PC) in lymphoid organs but, conversely, lower levels of plasmatic antibody titers. Exploring underlying mechanisms in human B cells revealed that exogenously added IL-38 did not significantly affect early B cell activation or differentiation into plasma cells, even though IL-38 suppressed upregulation of CD38. Instead, IL-38 mRNA expression was transiently upregulated during the differentiation of human B cells to plasma cells in vitro, and knocking down IL-38 during early B cell differentiation increased plasma cell generation, while reducing antibody production, thus reproducing the murine phenotype. Although this endogenous role of IL-38 in B cell differentiation and antibody production did not align with an immunosuppressive function, autoantibody production induced in mice by repeated IL-18 injections was enhanced in an IL-38-deficient background. Taken together, our data suggest that cell-intrinsic IL-38 promotes antibody production at baseline but suppresses the production of autoantibodies in an inflammatory context, which may partially explain its protective role during chronic inflammation.

Inhibition of IL-1ß release from macrophages targeted with necrosulfonamide-loaded porous nanoparticles.

Inflammation is required for protective responses against pathogens and is thus essential for survival, but sustained inflammation can lead to diseases, such as atherosclerosis and cancer. Two important mediators of inflammation are the cytokines IL-1ß and IL-18, which are produced by myeloid cells of the immune system, including macrophages. These cytokines are released into the extracellular space through pores formed in the plasma membrane by the oligomerized protein gasdermin D (GSDMD). Necrosulfonamide (NSA) was recently identified as an effective GSDMD inhibitor and represents a promising therapeutic agent in GSDMD-dependent inflammatory diseases. Here, we targeted NSA to both mouse and human macrophages by using three different types of porous nanoparticles (NP), i.e. mesoporous silica (MSN), porous crosslinked cyclodextrin carriers (CD-NP), and a mesoporous magnesium-phosphate carrier (MPC-NP), all displaying high loading capacities for this hydrophobic drug. Cellular uptake and intracellular NSA delivery were tracked in time-lapse experiments by live-cell, high-throughput fluorescence microscopy, demonstrating rapid nanoparticle uptake and effective targeted delivery of NSA to phagocytic cells. Notably, a strong cytostatic effect was observed when a macrophage cell line was exposed to free NSA. In contrast, cell growth was much less affected when NSA was delivered via the nanoparticle carriers. Utilizing NSA-loaded nanoparticles, a successful concentration-dependent suppression of IL-1ß secretion from freshly differentiated primary murine and human macrophages was observed. Functional assays showed the strongest suppressive effect on human macrophages when using CD-NP for NSA delivery, followed by MSN-NP. In contrast, MPC-NP completely blocked the metabolic activity in macrophages when loaded with NSA. This study demonstrates the potential of porous nanoparticles for the effective delivery of hydrophobic drugs to macrophages in order to suppress inflammatory responses.

Multifaceted roles of IL-38 in inflammation and cancer.

Interleukin (IL)-38 is the least well-understood cytokine of the IL-1 family. Since its discovery twenty years ago, numerous studies have linked IL-38 to diverse pathologies, especially in the context of autoimmune and inflammatory processes, while its role in cancer has been less explored. Broad anti-inflammatory effects have been reported for IL-38 in both in vitro and in vivo models, and, together with its homology to the IL-1 and IL-36 receptor antagonists, have raised expectations about its potential therapeutic utility. Data in human and mouse experimental systems support a negative regulatory role of IL-38 on the Th17 axis through effects on T cells and myeloid cells. Additional studies point to tolerogenic functions of IL-38, acting on dendritic cells and regulatory T cells, as well as to inhibition of pro-inflammatory macrophage activity. IL-38 further exhibits anti-inflammatory and tissue protective properties in epithelial and mesenchymal cells. However, published data also reveal variability and inconsistent dose-dependencies of these anti-inflammatory effects, as well as context-dependent pro-inflammatory properties of IL-38, and are difficult to interpret due to the high heterogeneity in the materials and experimental designs used across studies. In addition, it is still not clear which receptor(s) is/are fundamental for IL-38 signalling, and the biological impact of N-terminal processing of the protein remains to be clarified. In this review, we provide an overview of our current knowledge of IL-38 biology, discuss persistent controversies surrounding this cytokine, and highlight some questions to be addressed to facilitate progress towards a better understanding of its mechanisms of action.

3'mRNA sequencing reveals pro-regenerative properties of c5ar1 during resolution of murine acetaminophen-induced liver injury.

Murine acetaminophen-induced acute liver injury (ALI) serves as paradigmatic model for drug-induced hepatic injury and regeneration. As major cause of ALI, acetaminophen overdosing is a persistent therapeutic challenge with N-acetylcysteine clinically used to ameliorate parenchymal necrosis. To identify further treatment strategies that serve patients with poor N-acetylcysteine responses, hepatic 3'mRNA sequencing was performed in the initial resolution phase at 24 h/48 h after sublethal overdosing. This approach disclosed 45 genes upregulated (≥5-fold) within this time frame. Focusing on C5aR1, we observed in C5aR1-deficient mice disease aggravation during resolution of intoxication as evidenced by increased liver necrosis and serum alanine aminotransferase. Moreover, decreased hepatocyte compensatory proliferation and increased caspase-3 activation at the surroundings of necrotic cores were detectable in C5aR1-deficient mice. Using a non-hypothesis-driven approach, herein pro-regenerative/-resolving effects of C5aR1 were identified during late acetaminophen-induced ALI. Data concur with protection by the C5a/C5aR1-axis during hepatectomy and emphasize the complex role of inflammation during hepatic regeneration and repair.

Apoptotic Cells induce Proliferation of Peritoneal Macrophages.

The interaction of macrophages with apoptotic cells is required for efficient resolution of inflammation. While apoptotic cell removal prevents inflammation due to secondary necrosis, it also alters the macrophage phenotype to hinder further inflammatory reactions. The interaction between apoptotic cells and macrophages is often studied by chemical or biological induction of apoptosis, which may introduce artifacts by affecting the macrophages as well and/or triggering unrelated signaling pathways. Here, we set up a pure cell death system in which NIH 3T3 cells expressing dimerizable Caspase-8 were co-cultured with peritoneal macrophages in a transwell system. Phenotype changes in macrophages induced by apoptotic cells were evaluated by RNA sequencing, which revealed an unexpectedly dominant impact on macrophage proliferation. This was confirmed in functional assays with primary peritoneal macrophages and IC-21 macrophages. Moreover, inhibition of apoptosis during Zymosan-induced peritonitis in mice decreased mRNA levels of cell cycle mediators in peritoneal macrophages. Proliferation of macrophages in response to apoptotic cells may be important to increase macrophage numbers in order to allow efficient clearance and resolution of inflammation.

Inhibition of mPGES-1 attenuates efficient resolution of acute inflammation by enhancing CX3CL1 expression.

Despite the progress to understand inflammatory reactions, mechanisms causing their resolution remain poorly understood. Prostanoids, especially prostaglandin E2 (PGE2), are well-characterized mediators of inflammation. PGE2 is produced in an inducible manner in macrophages (Mϕ) by microsomal PGE2-synthase-1 (mPGES-1), with the notion that it also conveys pro-resolving properties. We aimed to characterize the role of mPGES-1 during resolution of acute, zymosan-induced peritonitis. Experimentally, we applied the mPGES-1 inhibitor compound III (CIII) once the inflammatory response was established and confirmed its potent PGE2-blocking efficacy. mPGES-1 inhibition resulted in an incomplete removal of neutrophils and a concomitant increase in monocytes and Mϕ during the resolution process. The mRNA-seq analysis identified enhanced C-X3-C motif receptor 1 (CX3CR1) expression in resident and infiltrating Mϕ upon mPGES-1 inhibition. Besides elevated Cx3cr1 expression, its ligand CX3CL1 was enriched in the peritoneal lavage of the mice, produced by epithelial cells upon mPGES-1 inhibition. CX3CL1 not only increased adhesion and survival of Mϕ but its neutralization also completely reversed elevated inflammatory cell numbers, thereby normalizing the cellular, peritoneal composition during resolution. Our data suggest that mPGES-1-derived PGE2 contributes to the resolution of inflammation by preventing CX3CL1-mediated retention of activated myeloid cells at sites of injury.