Parkinson’s disease (PD), the second most common neurodegenerative disorder after Alzheimer’s disease, and the rare disorder multiple system atrophy (MSA), are both characterized by intracellular accumulation of α-synuclein fibrils and early, sustained microglial reactivity in parallel to the neurodegeneration. Activation of the NLRP3 inflammasome in disease-associated reactive microglia is increasingly recognized as a key pathogenic driver and a promising therapeutic target in synucleinopathies. Dapansutrile (OLT1177®) is a selective, orally bioavailable NLRP3 inhibitor with a favorable safety profile in clinical trials for non-neurological indications. Here, we evaluated the therapeutic potential of dapansutrile in preclinical models of PD and MSA and explored the predictive and translational value of its effects. Two established mouse models of synucleinopathy with nigral neurodegeneration were employed: the α-synuclein preformed fibril (PFF) propagation model and the transgenic PLP-α-syn model expressing human wild-type α-synuclein in oligodendrocytes. Pharmacokinetic analyses assessed plasma and brain exposure after oral administration. The efficacy of six-month dapansutrile treatment was examined in both preventive (post-PFF injection) and therapeutic (PLP-α-syn mice) paradigms, using behavioral, histopathological, and molecular readouts. Transcriptomic profiling of striatal and midbrain microglia identified differentially expressed genes (DEGs) associated with treatment and compared them with post-mortem transcriptomic signatures of disease-associated microglia in PD patients. Plasma IL-18 and neurofilament light chain (NfL) levels were evaluated as translational biomarkers. Chronic oral dapansutrile treatment at clinically relevant doses improved motor performance, reduced α-synuclein inclusions, attenuated gliosis, and mitigated nigral neurodegeneration in both models. Microglial transcriptomic analyses revealed that dapansutrile reversed key transcriptional signatures characteristic of PD-associated reactive microglia. Moreover, plasma IL-18 and NfL levels correlated with neuropathological and functional outcomes, supporting their potential as biomarkers of target engagement and treatment efficacy. These data identify chronic NLRP3 activation as a shared and targetable mechanism in PD and MSA and highlight dapansutrile as a CNS-penetrant, clinically advanced candidate for disease modification in α-synucleinopathies. The observed transcriptomic reprogramming of microglia and the parallel changes in blood biomarkers provide a strong translational bridge to clinical development.

Background Parkinson’s disease (PD), the second most common neurodegenerative disorder after Alzheimer’s disease, and the rare disorder multiple system atrophy (MSA), are both characterized by intracellular accumulation of α-synuclein fibrils and early, sustained microglial reactivity in parallel to the neurodegeneration. Activation of the NLRP3 inflammasome in disease-associated reactive microglia is increasingly recognized as a key pathogenic driver and a promising therapeutic target in synucleinopathies. Dapansutrile (OLT1177®) is a selective, orally bioavailable NLRP3 inhibitor with a favorable safety profile in clinical trials for non-neurological indications. Here, we evaluated the therapeutic potential of dapansutrile in preclinical models of PD and MSA and explored the predictive and translational value of its effects. Methods Two established mouse models of synucleinopathy with nigral neurodegeneration were employed: the α-synuclein preformed fibril (PFF) propagation model and the transgenic PLP-α-syn model expressing human wild-type α-synuclein in oligodendrocytes. Pharmacokinetic analyses assessed plasma and brain exposure after oral administration. The efficacy of six-month dapansutrile treatment was examined in both preventive (post-PFF injection) and therapeutic (PLP-α-syn mice) paradigms, using behavioral, histopathological, and molecular readouts. Transcriptomic profiling of striatal and midbrain microglia identified differentially expressed genes (DEGs) associated with treatment and compared them with post-mortem transcriptomic signatures of disease-associated microglia in PD patients. Plasma IL-18 and neurofilament light chain (NfL) levels were evaluated as translational biomarkers. Results Chronic oral dapansutrile treatment at clinically relevant doses improved motor performance, reduced α-synuclein inclusions, attenuated gliosis, and mitigated nigral neurodegeneration in both models. Microglial transcriptomic analyses revealed that dapansutrile reversed key transcriptional signatures characteristic of PD-associated reactive microglia. Moreover, plasma IL-18 and NfL levels correlated with neuropathological and functional outcomes, supporting their potential as biomarkers of target engagement and treatment efficacy. Conclusions These data identify chronic NLRP3 activation as a shared and targetable mechanism in PD and MSA and highlight dapansutrile as a CNS-penetrant, clinically advanced candidate for disease modification in α-synucleinopathies. The observed transcriptomic reprogramming of microglia and the parallel changes in blood biomarkers provide a strong translational bridge to clinical development. ### Competing Interest Statement ML, declares no competing interests MM, declares no competing interests AHG, declares no competing interests JAA, declares no competing interests KCL, declares no competing interests MTH, declares no competing interests MK, has received travel funding and speaker honoraria from Bayer, Biogen, Novartis, Merck, Sanofi, Roche and Teva, serves on scientific advisory boards for Biogen, Bristol-Myers Squibb, Gilead, Merck, Neuraxpharm, Novartis, Alexion, Amgen and Roche and as a consultant for Roche. He received research grants from the Austrian MS Society, Biogen, Novartis and Roche. DBS, Chief Executive Officer of Olatec Therapeutics CAD, Chairman of Olatec's Scientific Advisory Board, co-Chief Scientific Officer, receives compensation, and has equity in Olatec NS, Director-at-large, Board of Directors and Chair of the Research Steering Council of Mission MSA, USA; Advisory board, Karl Golser Foundation, Italy; Advisory services for IONIS, Mitsubishi Pharma * 6-OHDA : 6-hydroxydopamine CNS : central nervous system DEGs : differentially expressed genes MPTP : 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine MSA : multiple system atrophy NfL : neurofilament light chain NLRP3 : NOD-, LRR-, and pyrin domain-containing protein 3 PD : Parkinson’s disease PFF : pre-formed fibrils PK : pharmacokinetic PLP : proteolipid protein pS129 : hyperphosphorylated serine 129 α-synuclein SNc : substantia nigra pars compacta α-syn : α-synuclein Michael J. Fox Foundation, https://ror.org/03arq3225, MJFF-022681

IntroductionInterleukin-18 (IL-18), a pro-inflammatory cytokine belonging to the IL-1 Family, is a key mediator ofautoinflammatory diseases associated with the development of macrophage activation syndrome (MAS).High levels of IL-18 correlate with MAS and COVID-19 severity and mortality, particularly in COVID-19patients with MAS. As an inflammation inducer, IL-18 binds its receptor IL-1 Receptor 5 (IL-1R5), leadingto the recruitment of the co-receptor, IL-1 Receptor 7 (IL-1R7). This heterotrimeric complex subsequentlyinitiates downstream signaling, resulting in local and systemic inflammation.MethodsWe reported earlier the development of a novel humanized monoclonal anti-human IL-1R7 antibody whichspecifically blocks the activity of human IL-18 and its inflammatory signaling in human cell and wholeblood cultures. In the current study, we further explored the strategy of blocking IL-1R7 inhyperinflammation in vivo using animal models.ResultsWe first identified an anti-mouse IL-1R7 antibody that significantly suppressed mouse IL-18 andlipopolysaccharide (LPS)-induced IFNg production in mouse splenocyte and peritoneal cell cultures. Whenapplied in vivo, the antibody reduced Propionibacterium acnes and LPS-induced liver injury and protectedmice from tissue and systemic hyperinflammation. Importantly, anti-IL-1R7 significantly inhibited plasma,liver cell and spleen cell IFNg production. Also, anti-IL-1R7 downregulated plasma TNFa, IL-6, IL-1b,MIP-2 production and the production of the liver enzyme ALT. In parallel, anti-IL-1R7 suppressed LPSinducedinflammatory cell infiltration in lungs and inhibited the subsequent IFNg production andinflammation in mice when assessed using an acute lung injury model.DiscussionAltogether, our data suggest that blocking IL-1R7 represents a potential therapeutic strategy to specificallymodulate IL-18-mediated hyperinflammation, warranting further investigation of its clinical application intreating IL-18-mediated diseases, including MAS and COVID-19.

NLRP3 activation mediates a Th2-like inflammatory response. A, Flow cytometry analysis of CD4, CD4/PD1, CD4/CD44/PD-1 cells in primary tumors of mice fed standard (STD) and OLT1177 (OLT)-enriched diets (n = 5/group). B, Flow cytometry analysis of memory (CD62L+/CD44+), naïve (CD62L+/CD44−), and effector (CD62L−/CD44+) CD4 cells in primary tumors of mice fed STD and OLT-enriched diets (n = 5/group). C, Gene expression of Il2, Il4, and Il10 in primary tumors of mice of mice fed STD and OLT-enriched diets (n = 5/group). D, COX-2 and PGE2 levels measured by ELISA in primary tumors of mice of mice fed STD and OLT-enriched diets (n = 5/group). E, PGE2 levels measured by ELISA in primary tumors of mice of mice fed STD and OLT-enriched diets (n = 5/group). Data expressed as mean ± SEM; ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05.

NLRP3 inhibition reduces PDAC progression

Comparison of tumor growth in Nlrp3-/- mice treated with OLT1177

NLRP3 expression and activation are increased in PDAC biopsy. A, NLRP3, ASC, IL1β, and IL18 median expression levels in human PDAC biopsy and normal pancreas samples (TCGA dataset). B, Immunofluorescence staining from NLRP3 (red) and ASC (green) in biopsy of normal pancreas (n = 2) and patients with PDAC (n = 4). Each row represents a single patient. C, Quantification of ASC specks–like structure samples in B. *, P < 0.05.

Metabolism is a fundamental process by which biochemicals are broken down to produce energy (catabolism) or used to build macromolecules (anabolism). Metabolism has received renewed attention as a mechanism that generates molecules that modulate multiple cellular responses. This was first identified in cancer cells as the Warburg effect, but it is also present in immunocompetent cells. Studies have revealed a bidirectional influence of cellular metabolism and immune cell function, highlighting the significance of metabolic reprogramming in immune cell activation and effector functions. Metabolic processes such as glycolysis, oxidative phosphorylation, and fatty acid oxidation have been shown to undergo dynamic changes during immune cell response, facilitating the energetic and biosynthetic demands. This review aims to provide a better understanding of the metabolic reprogramming that occurs in different immune cells upon activation, with a special focus on central nervous system disorders. Understanding the metabolic changes of the immune response not only provides insights into the fundamental mechanisms that regulate immune cell function but also opens new approaches for therapeutic strategies aimed at manipulating the immune system.

CSF-1R engagement induces NLRP3 activation in vivo. A, Tumor volume and weight in mice treated with α-CSF-1R or matching IgG (n = 6/group). B, Immunofluorescence staining from NLRP3 (red) and ASC (green) in primary tumors treated with α-CSF-1R or matching IgG (n = 3/group). C, Quantification of speck-like structure in mice treated with α-CSF-1R or matching IgG (n = 3/group). D, Schematic graphic representation of the proposed NLRP3 activation and signaling in PDAC (created with BioRender.com).

Gating strategy used for the definition of the populations of interest.

Defining feature of pancreatic ductal adenocarcinoma (PDAC) that participates in the high mortality rate and drug resistance is the immune-tolerant microenvironment which enables tumors to progress unabated by adaptive immunity. In this study, we report that PDAC cells release CSF-1 to induce nucleotide-binding domain, leucine-rich containing family, pyrin domain-containing-3 (NLRP3) activation in myeloid cells. Increased NLRP3 expression was found in the pancreas of patients with PDAC when compared with normal pancreas which correlated with the formation of the NLRP3 inflammasome. Using human primary cells and an orthotopic PDAC mouse model, we show that NLRP3 activation is responsible for the maturation and release of the inflammatory cytokine IL1β which selectively drives Th2-type inflammation via COX2/PGE2 induction. As a result of this inflammation, primary tumors were characterized by reduced cytotoxic CD8+ T-cell activation and increased tumor expansion. Genetic deletion and pharmacologic inhibition of NLRP3 enabled the development of Th1 immunity, increased intratumoral levels of IL2, CD8+ T cell–mediated tumor suppression, and ultimately limited tumor growth. In addition, we observed that NLRP3 inhibition in combination with gemcitabine significantly increased the efficacy of the chemotherapy. In conclusion, this study provides a mechanism by which tumor-mediated NLRP3 activation exploits a distinct adaptive immunity response that facilitates tumor escape and progression. Considering the ability to block NLRP3 activity with safe and small orally active molecules, this protein represents a new promising target to improve the limited therapeutic options in PDAC. Significant: This study provides novel molecular insights on how PDAC cells exploit NLRP3 activation to suppress CD8 T-cell activation. From a translational perspective, we demonstrate that the combination of gemcitabine with the orally active NLRP3 inhibitor OLT1177 increases the efficacy of monotherapy.

The IL-1 Family member IL-38 has been characterized primarily as an antiinflammatory cytokine in human and mouse models of systemic diseases. Here, we examined the role of IL-38 in the murine small intestine (SI). Immunostaining of SI revealed that IL-38 expression partially confines to intestinal stem cells. Cultures of intestinal organoids reveal IL-38 functions as a growth factor by increasing organoid size via inducing WNT3a. In contrast, organoids from IL-38-deficient mice develop more slowly. This reduction in size is likely due to the downregulation of intestinal stemness markers (i.e., Fzd5 , Ephb2 , and Olfm4 ) expression compared with wild-type organoids. The IL-38 binding to IL-1R6 and IL-1R9 is still a matter of debate. Therefore, to analyze the molecular mechanisms of IL-38 signaling, we also examined organoids from IL-1R9-deficient mice. Unexpectedly, these organoids, although significantly smaller than wild type, respond to IL-38, suggesting that IL-1R9 is not involved in IL-38 signaling in the stem cell crypt. Nevertheless, silencing of IL-1R6 disabled the organoid response to the growth property of IL-38, thus suggesting IL-1R6 as the main receptor used by IL-38 in the crypt compartment. In organoids from wild-type mice, IL-38 stimulation induced low concentrations of IL-1β which contribute to organoid growth. However, high concentrations of IL-1β have detrimental effects on the cultures that were prevented by treatment with recombinant IL-38. Overall, our data demonstrate an important regulatory function of IL-38 as a growth factor, and as an antiinflammatory molecule in the SI, maintaining homeostasis.

CSF-1R engagement induces IL1β and IL18 release. A, Correlation between CSF-1R and NLRP3 expression in PDAC (n = 96) from TGCA dataset. B, Spontaneous CSF-1 and IL34 secretion in Panc-1 cells at 72 hours (n = 3). C, IL1β and IL18 secretion in THP-1 cells following stimulation with PDAC-conditioned media obtained from Panc-1 cells in presence of IgG (control), α-CSF-1, α-IL34, α-CSF-1/IL34 (n = 3). D, Phosphorylation levels of PyK2 (Tyrosine 402) in THP-1 stimulated in D and E (n = 3). E, IL1β and IL18 secretion from THP-1 cells following stimulation with PDAC-conditioned media obtained from Panc-1 cells in presence of α-CSF-1R and an CSF-1R inhibitor (AZD; n = 3). F, IL1β secretion from freshly isolated PBMCs from healthy donors (n = 4) following stimulation with PDAC-conditioned media obtained from Panc-1 cells in presence of α-CSF-1R. Cytokine levels were measured by specific ELISAs. Data expressed as mean ± SEM; ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05.

Background Parkinson’s disease (PD) is characterized by a progressive degeneration of dopaminergic neurons, which leads to irreversible loss of peripheral motor functions. Death of dopaminergic neurons induces an inflammatory response in microglial cells, which further exacerbates neuronal loss. Reducing inflammation is expected to ameliorate neuronal loss and arrest motor dysfunctions. Because of the contribution of the NLRP3 inflammasome to the inflammatory response in PD, we targeted NLRP3 using the specific inhibitor OLT1177 ® . Methods We evaluated the effectiveness of OLT1177 ® in reducing the inflammatory response in an MPTP neurotoxic model of PD. Using a combination of in vitro and in vivo studies, we analyzed the effects of NLRP3 inhibition on pro-inflammatory markers in the brain, α-synuclein aggregation, and dopaminergic neuron survival. We also determined the effects of OLT1177 ® on locomotor deficits associated with MPTP and brain penetrance. Results Treatment with OLT1177 ® prevented the loss of motor function, reduced the levels of α-synuclein, modulated pro-inflammatory markers in the nigrostriatal areas of the brain, and protected dopaminergic neurons from degeneration in the MPTP model of PD. We also demonstrated that OLT1177 ® crosses the blood–brain barrier and reaches therapeutic concentrations in the brain. Conclusions These data suggest that targeting the NLRP3 inflammasome by OLT1177 ® may be a safe and novel therapeutic approach to arrest neuroinflammation and protect against neurological deficits of Parkinson’s disease in humans.

NLRP3 activation mediates lympthocyte suppression. A, Tumor volume and weight in NCG mice fed standard (STD) or OL1177 (OLT) diet (n = 5/group). B, COX-2 levels measured by ELISAs in primary tumors of mice of mice fed STD and OLT-enriched diets. C, PGE2 levels measured by ELISAs in primary tumors of mice of mice fed STD and OLT-enriched diets. Data expressed as mean ± SEM. *, P < 0.05.

Level of NLRP3, IL 1b and IL 18 in PDAC cells.

IL-38 is a recently discovered cytokine and member of the IL-1 Family. In the IL-1 Family, IL-38 is unique because the cytokine is primarily a B lymphocyte product and functions to suppress inflammation. Studies in humans with inflammatory bowel disease (IBD) suggest that IL-38 may be protective for ulcerative colitis or Crohn’s disease, and that IL-38 acts to maintain homeostasis in the intestinal tract. Here we investigated the role of endogenous IL-38 in experimental colitis in mice deficient in IL-38 by deletion of exons 1-4 in C57 BL/6 mice. Compared to WT mice, IL-38 deficient mice subjected to dextran sulfate sodium (DSS) showed greater severity of disease, more weight loss, increased intestinal permeability, and a worse histological phenotype including increased neutrophil influx in the colon. Mice lacking IL-38 exhibited elevated colonic Nlrp3 mRNA and protein levels, increased caspase-1 activation, and the concomitant increased processing of IL-1β precursor into active IL-1β. Expression of IL-1α, an exacerbator of IBD, was also upregulated. Colonic myleloperoxidase protein and Il17a, and Il17f mRNA levels were higher in the IL-38 deficient mice. Daily treatment of IL-38 deficient mice with an NLRP3 inhibitor attenuated diarrhea and weight loss during the recovery phase. These data implicate endogenous IL-38 as an anti-inflammatory cytokine that reduces DSS colitis severity. We propose that a relative deficiency of IL-38 contributes to IBD by disinhibition of the NLRP3 inflammasome.

Cytokines play crucial roles in the pathophysiology after spinal cord injury (SCI). After SCI, there is an activation of resident glial cells such as microglia and astrocytes, as well as an infiltration of blood immune cells. These cells produce sequential segregation of huge amounts of cytokines which stimulate diverse processes. Thus, the proinflammatory and proregenerative actions of these molecules impact on the functional outcome and recovery after a neural trauma. The aim of the present book chapter is to describe the role of cytokines within the progression after the injury as well as to describe the main research findings that envisage them as therapeutic targets to modulate neurodegeneration, promote repair, and improve functional outcomes after SCI.

Activating and inhibitory immune receptors play a critical role in regulating systemic and central nervous system (CNS) immune and inflammatory processes. The CD200R1 immunoreceptor induces a restraining signal modulating inflammation and phagocytosis in the CNS under different inflammatory conditions. However, it remains unknown whether CD200R1 has a role in modulating the inflammatory response after a peripheral nerve injury, an essential component of the successful regeneration. Expression of CD200R1 and its ligand CD200 was analyzed during homeostasis and after a sciatic nerve crush injury in C57Bl/6 mice. The role of CD200R1 in Wallerian Degeneration (WD) and nerve regeneration was studied using a specific antibody against CD200R1 injected into the nerve at the time of injury. We found an upregulation of CD200R1 mRNA after injury whereas CD200 was downregulated acutely after nerve injury. Blockade of CD200R1 significantly reduced the acute entrance of both neutrophils and monocytes from blood after nerve injury. When long term regeneration and functional recovery were evaluated, we found that blockade of CD200R1 had a significant effect impairing the spontaneous functional recovery. Taken together, these results show that CD200R1 has a role in mounting a successful acute inflammatory reaction after injury, and contributes to an effective functional recovery.