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Pancreatic cancer is characterized by extreme therapeutic resistance. In pancreatic cancers harboring high-risk genomes, we describe that cancer cell-neutrophil signaling circuitry provokes neutrophil-derived transmembrane (tm)TNF-TNFR2 interactions that dictate inflammatory polarization in cancer-associated fibroblasts and T-cell dysfunction – two hallmarks of therapeutic resistance. Targeting tmTNF-TNFR2 signaling may sensitize pancreatic cancer to chemo±immunotherapy.

Abstract Background Preterm birth is often associated with chorioamnionitis and leads to increased risk of neurodevelopmental disorders, such as autism. Preterm birth can lead to cerebellar underdevelopment, but the mechanisms of disrupted cerebellar development in preterm infants are not well understood. The cerebellum is consistently affected in people with autism spectrum disorders, showing reduction of Purkinje cells, decreased cerebellar grey matter, and altered connectivity. Methods Preterm rhesus macaque fetuses were exposed to intra-amniotic LPS (1 mg, E. coli O55:B5) at 127 days (80%) gestation and delivered by c-section 5 days after injections. Maternal and fetal plasma were sampled for cytokine measurements. Chorio-decidua was analyzed for immune cell populations by flow cytometry. Fetal cerebellum was sampled for histology and molecular analysis by single-nuclei RNA-sequencing (snRNA-seq) on a 10× chromium platform. snRNA-seq data were analyzed for differences in cell populations, cell-type specific gene expression, and inferred cellular communications. Results We leveraged snRNA-seq of the cerebellum in a clinically relevant rhesus macaque model of chorioamnionitis and preterm birth, to show that chorioamnionitis leads to Purkinje cell loss and disrupted maturation of granule cells and oligodendrocytes in the fetal cerebellum at late gestation. Purkinje cell loss is accompanied by decreased sonic hedgehog signaling from Purkinje cells to granule cells, which show an accelerated maturation, and to oligodendrocytes, which show accelerated maturation from pre-oligodendrocytes into myelinating oligodendrocytes. Conclusion These findings suggest a role of chorioamnionitis on disrupted cerebellar maturation associated with preterm birth and on the pathogenesis of neurodevelopmental disorders among preterm infants.

In central nervous system (CNS) injury and disease, peripherally derived myeloid cells infiltrate the CNS parenchyma and interact with resident cells, propagating the neuroinflammatory response. Because peripheral myeloid populations differ profoundly depending on the type and phase of injury, their crosstalk with CNS resident cells, particularly microglia, will lead to different functional outcomes. Thus, understanding how peripheral myeloid cells affect the phenotype and function of microglia in different disease conditions and phases may lead to a better understanding of disease-specific targetable pathways for neuroprotection and neurorepair. To this end, we set out to develop an in vitro system to investigate the communication between peripheral myeloid cells and microglia, with the goal of uncovering potential differences due to disease type and timing. We isolated peripheral myeloid cells from mice undergoing experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis, or acute cerebral ischemia by permanent middle cerebral artery occlusion (pMCAO) at different times after disease and probed their ability to change the phenotype of primary microglia isolated from the brain of adult mice. We identified changes not only dependent on the disease model, but also on the timepoint after disease onset from which the myeloid cells were isolated. Peripheral myeloid cells from acute EAE induced morphological changes in microglia, followed by increases in expression of genes involved in inflammatory signaling. Conversely, it was the peripheral myeloid cells from the chronic phase of pMCAO that induced gene expression changes in genes involved in inflammatory signaling and phagocytosis, which was not followed by a change in morphology. This underscores the importance of understanding the role of infiltrating myeloid cells in different disease contexts and phases. Furthermore, we showed that our assay is a valuable tool for investigating myeloid cell interactions in a range of CNS neuroinflammatory conditions.

Abstract Background Mechanical ventilation of preterm newborns causes lung injury and is associated with poor neurodevelopmental outcomes. However, the mechanistic links between ventilation-induced lung injury (VILI) and brain injury is not well defined. Since circulating extracellular vesicles (EVs) are known to link distant organs by transferring their cargos, we hypothesized that EVs mediate inflammatory brain injury associated with VILI. Methods Neonatal rats were mechanically ventilated with low (10 mL/kg) or high (25 mL/kg) tidal volume for 1 h on post-natal day 7 followed by recovery for 2 weeks. Exosomes were isolated from the plasma of these rats and adoptively transferred into normal newborn rats. We assessed the effect of mechanical ventilation or exosome transfer on brain inflammation and activation of the pyroptosis pathway by western blot and histology. Results Injurious mechanical ventilation induced similar markers of inflammation and pyroptosis, such as increased IL-1β and activated caspase-1/gasdermin D (GSDMD) in both lung and brain, in addition to inducing microglial activation and cell death in the brain. Isolated EVs were enriched for the exosomal markers CD9 and CD81, suggesting enrichment for exosomes. EVs isolated from neonatal rats with VILI had increased caspase-1 but not GSDMD. Adoptive transfer of these EVs led to neuroinflammation with microglial activation and activation of caspase-1 and GSDMD in the brain similar to that observed in neonatal rats that were mechanically ventilated. Conclusions These findings suggest that circulating EVs can contribute to the brain injury and poor neurodevelopmental outcomes in preterm infants with VILI through activation of GSDMD.

Multiple sclerosis (MS) is the most common neurological disorder in young adults and is classically defined as a chronic inflammatory demyelinating disease of the central nervous system (CNS). Although MS affects millions of people worldwide, its underlying cause remains unknown making discovery of effective treatments challenging. Whether intrinsic or extrinsic factors contribute to MS initiation and progression is still unclear. This is especially true for primary progressive MS (PPMS), the rarest form of the disease, in which progressive and irreversible loss of neurological function is often observed in the absence of an overt immune-inflammatory response. To test the hypothesis that intrinsic dysfunction in oligodendrocytes (OLs), the primary targets of damage in MS, may contribute to PPMS etiopathology, we differentiated human induced pluripotent stem cell (hiPSC) lines derived from PPMS and healthy individuals into mature OLs to compare their transcriptional profile. PPMS derived OLs displayed hundreds of differentially expressed genes compared to control OLs, many associated with cell adhesion, apoptosis and inflammation, including the inflammasome component Nlrp2, which was highly upregulated. NLRP2 immunoreactivity in OLs was confirmed in post-mortem PPMS brain tissues, with higher expression than in control tissues. Altogether, our findings suggest that mature OLs in PPMS affected individuals carry intrinsic abnormalities that could contribute, at least in part, to the pathophysiology of this form of the disease.

Clinical and animal model studies have implicated inflammation and glial and peripheral immune cell responses in the pathophysiology of spinal cord injury (SCI). A key player in the inflammatory response after SCI is the pleiotropic cytokine tumor necrosis factor (TNF), which exists both in both a transmembrane (tmTNF) and a soluble (solTNF) form. In the present study, we extend our previous findings of a therapeutic effect of topically blocking solTNF signaling after SCI for three consecutive days on lesion size and functional outcome to study the effect on spatio-temporal changes in the inflammatory response after SCI in mice treated with the selective solTNF inhibitor XPro1595 and compared to saline-treated mice. We found that despite comparable TNF and TNF receptor levels between XPro1595- and saline-treated mice, XPro1595 transiently decreased pro-inflammatory interleukin (IL)-1β and IL-6 levels and increased pro-regenerative IL-10 levels in the acute phase after SCI. This was complemented by a decrease in the number of infiltrated leukocytes (macrophages and neutrophils) in the lesioned area of the spinal cord and an increase in the number of microglia in the peri-lesion area 14 days after SCI, followed by a decrease in microglial activation in the peri-lesion area 21 days after SCI. This translated into increased myelin preservation and improved functional outcomes in XPro1595-treated mice 35 days after SCI. Collectively, our data suggest that selective targeting of solTNF time-dependently modulates the neuroinflammatory response by favoring a pro-regenerative environment in the lesioned spinal cord, leading to improved functional outcomes.

Abstract Background The inflammasome adaptor apoptosis-associated speck-like protein containing a CARD (ASC) is involved in immune signaling by bridging the interactions between inflammasome sensors and caspase-1. Strong experimental evidence has shown that ASC−/− mice are protected from disease progression in animal models of multiple sclerosis (MS), suggesting that targeting inflammasome activation via ASC inhibition may be a promising therapeutic strategy in MS. Thus, the goal of our study is to test the efficacy of IC100, a novel humanized antibody targeting ASC, in preventing and/or suppressing disease in the experimental autoimmune encephalomyelitis (EAE) model of MS. Methods We employed the EAE model of MS where disease was induced by immunization of C57BL/6 mice with myelin oligodendrocyte glycoprotein peptide 35–55 (MOG35–55). Mice were treated with vehicle or increasing doses of IC100 (10, 30, and 45 mg/kg) and clinical disease course was evaluated up to 35 days post EAE induction. Immune cell infiltration into the spinal cord and microglia responses were assessed. Results We show that IC100 treatment reduced the severity of EAE when compared to vehicle-treated controls. At a dose of 30 mg/kg, IC100 significantly reduced the number of CD4+ and CD8+ T cells and CD11b+MHCII+ activated myeloid cells entering the spinal cord from the periphery, and reduced the number of total and activated microglia. Conclusions These data indicate that IC100 suppresses the immune-inflammatory response that drives EAE development and progression, thereby identifying ASC as a promising target for the treatment of MS as well as other neurological diseases with a neuroinflammatory component.

Spinal cord injury (SCI) initiates detrimental cellular and molecular events that lead to acute and delayed neuroinflammation. Understanding the role of the inflammatory response in SCI requires insight into the temporal and cellular synthesis of inflammatory mediators. We subjected C57BL/6J mice to SCI and investigated inflammatory reactions. We examined activation, recruitment, and polarization of microglia and infiltrating immune cells, focusing specifically on tumor necrosis factor (TNF) and its receptors TNFR1 and TNFR2. In the acute phase, TNF expression increased in glial cells and neuron-like cells, followed by infiltrating immune cells. TNFR1 and TNFR2 levels increased in the delayed phase and were found preferentially on neurons and glial cells, respectively. The acute phase was dominated by the infiltration of granulocytes and macrophages. Microglial/macrophage expression of Arg1 increased from 1–7 days after SCI, followed by an increase in Itgam, Cx3cr1, and P2ry12, which remained elevated throughout the study. By 21 and 28 days after SCI, the lesion core was populated by galectin-3+, CD68+, and CD11b+ microglia/macrophages, surrounded by a glial scar consisting of GFAP+ astrocytes. Findings were verified in postmortem tissue from individuals with SCI. Our findings support the consensus that future neuroprotective immunotherapies should aim to selectively neutralize detrimental immune signaling while sustaining pro-regenerative processes.

Microglia play an essential role in maintaining central nervous system (CNS) homeostasis, as well as responding to injury and disease. Most neurological disorders feature microglial activation, a process whereby microglia undergo profound morphological and transcriptional changes aimed at containing CNS damage and promoting repair, but often resulting in overt inflammation that sustains and propagates the neurodegenerative process. This is especially evident in multiple sclerosis (MS), were microglial activation and microglia-driven neuroinflammation are considered key events in the onset, progression, and resolution of the disease. Our understanding of microglial functions in MS has widened exponentially in the last decade by way of new tools and markers to discriminate microglia from other myeloid populations. Consequently, the complex functional and phenotypical diversity of microglia can now be appreciated. This, in combination with a variety of animal models that mimic specific features and processes of MS, has contributed to filling the gap of knowledge in the cascade of events underlying MS pathophysiology. The purpose of this review is to present the most up to date knowledge of the dynamic responses of microglia in the commonly used animal models of MS, specifically the immune-mediated experimental autoimmune encephalomyelitis (EAE) model, and the chemically-induced cuprizone and lysolecithin models. Elucidating the spectrum of microglial functions in these models, from detrimental to protective, is essential to identify emerging targets for therapy and guide drug discovery efforts.

Remyelination failure is a crucial component of disease progression in the autoimmune demyelinating disease Multiple Sclerosis (MS). The regenerative capacity of oligodendrocyte progenitor cells (OPCs) to replace myelinating oligodendrocytes is likely influenced by many aspects of the lesion environment including inflammatory signaling and extracellular matrix (ECM) deposition. These features of MS lesions are typically attributed to infiltrating leukocytes and reactive astrocytes. Here we demonstrate that fibroblasts also contribute to the inhibitory environment in the animal model of MS, experimental autoimmune encephalomyelitis (EAE). Using Col1α1GFP transgenic mice, we show that perivascular fibroblasts are activated in the spinal cord at EAE onset, and infiltrate the parenchyma by the peak of behavioral deficits where they are closely associated with areas of demyelination, myeloid cell accumulation, and ECM deposition. We further show that both fibroblast conditioned media and fibroblast ECM inhibit the differentiation of OPCs into mature oligodendrocytes. Taken together, our results indicate that the fibrotic scar is a major component of EAE pathology that leads to an inhibitory environment for remyelination, thus raising the possibility that anti-fibrotic mechanisms may serve as novel therapeutic targets for MS.

Multiple sclerosis (MS) is a neuroimmune disorder characterized by inflammation, CNS demyelination, and progressive neurodegeneration. Chronic MS patients exhibit impaired remyelination capacity, partly due to the changes that oligodendrocyte precursor cells (OPCs) undergo in response to the MS lesion environment. The cytokine tumor necrosis factor (TNF) is present in the MS-affected CNS and has been implicated in disease pathophysiology. Of the two active forms of TNF, transmembrane (tmTNF) and soluble (solTNF), tmTNF signals via TNFR2 mediating protective and reparative effects, including remyelination, whereas solTNF signals predominantly via TNFR1 promoting neurotoxicity. To better understand the mechanisms underlying repair failure in MS, we investigated the cellular responses of OPCs to inflammatory exposure and the specific role of TNFR2 signaling in their modulation. Following treatment of cultured OPCs with IFNγ, IL1β, and TNF, we observed, by RNA sequencing, marked inflammatory and immune activation of OPCs, accompanied by metabolic changes and dysregulation of their proliferation and differentiation programming. We also established the high likelihood of cell–cell interaction between OPCs and microglia in neuroinflammatory conditions, with OPCs able to produce chemokines that can recruit and activate microglia. Importantly, we showed that these functions are exacerbated when TNFR2 is ablated. Together, our data indicate that neuroinflammation leads OPCs to shift towards an immunomodulatory phenotype while diminishing their capacity to proliferate and differentiate, thus impairing their repair function. Furthermore, we demonstrated that TNFR2 plays a key role in this process, suggesting that boosting TNFR2 activation or its downstream signals could be an effective strategy to restore OPC reparative capacity in demyelinating disease.

BackgroundTumor necrosis factor, which exists both as a soluble (solTNF) and a transmembrane (tmTNF) protein, plays an important role in post-stroke inflammation. The objective of the present study was to test the effect of topical versus intracerebroventricular administration of XPro1595 (a solTNF inhibitor) and etanercept (a solTNF and tmTNF inhibitor) compared to saline on output measures such as infarct volume and post-stroke inflammation in mice.MethodsAdult male C57BL/6 mice were treated topically (2.5 mg/ml/1μl/h for 3 consecutive days) or intracerebroventricularly (1.25 mg/kg/0.5 ml, once) with saline, XPro1595, or etanercept immediately after permanent middle cerebral artery occlusion (pMCAO). Mice were allowed to survive 1 or 3 days. Infarct volume, microglial and leukocyte profiles, and inflammatory markers were evaluated.ResultsWe found that topical, and not intracerebroventricular, administration of XPro1595 reduced infarct volume at both 1 and 3 days after pMCAO. Etanercept showed no effect. We observed no changes in microglial or leukocyte populations. XPro1595 increased gene expression of P2ry12 at 1 day and Trem2 at 1 and 3 days, while decreasing Cx3cr1 expression at 1 and 3 days after pMCAO, suggesting a change in microglial activation toward a phagocytic phenotype.ConclusionOur data demonstrate that topical administration of XPro1595 for 3 consecutive days decreases infarct volumes after ischemic stroke, while modifying microglial activation and the inflammatory response post-stroke. This suggests that inhibitors of solTNF hold great promise for future neuroprotective treatment in ischemic stroke.

Spinal cord injury (SCI) is a devastating condition consisting of an instant primary mechanical injury followed by a secondary injury that progresses for weeks to months. The cytokine tumor necrosis factor (TNF) plays an important role in the pathophysiology of SCI. We investigated the effect of myeloid TNF ablation (peripheral myeloid cells (macrophages and neutrophils) and microglia) versus central myeloid TNF ablation (microglia) in a SCI contusion model. We show that TNF ablation in macrophages and neutrophils leads to reduced lesion volume and improved functional outcome after SCI. In contrast, TNF ablation in microglia only or TNF deficiency in all cells had no effect. TNF levels tended to be decreased 3 h post-SCI in mice with peripheral myeloid TNF ablation and was significantly decreased 3 days after SCI. Leukocyte and microglia populations and all other cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, and IFNγ) and chemokines (CCL2, CCL5, and CXCL1) investigated, in addition to TNFR1 and TNFR2, were comparable between genotypes. Analysis of post-SCI signaling cascades demonstrated that the MAPK kinase SAPK/JNK decreased and neuronal Bcl-XL levels increased post-SCI in mice with ablation of TNF in peripheral myeloid cells. These findings demonstrate that peripheral myeloid cell-derived TNF is pathogenic in SCI.

Traumatic spinal cord injury (SCI) is followed by an instant increase in expression of the microglial-derived proinflammatory cytokine tumor necrosis factor (TNF) within the lesioned cord. TNF exists both as membrane-anchored TNF (mTNF) and as cleaved soluble TNF (solTNF). We previously demonstrated that epidural administration of a dominant-negative inhibitor of solTNF, XPro1595, to the contused spinal cord resulted in changes in Iba1 protein expression in microglia/macrophages, decreased lesion volume, and improved locomotor function. Here, we extend our studies using mice expressing mTNF, but no solTNF (mTNFΔ/Δ), to study the effect of genetic ablation of solTNF on SCI. We demonstrate that TNF levels were significantly decreased within the lesioned spinal cord 3 days after SCI in mTNFΔ/Δ mice compared to littermates. This decrease did, however, not translate into significant changes in other pro- and anti-inflammatory cytokines (IL-10, IL-1β, IL-6, IL-5, IL-2, CXCL1, CCL2, or CCL5), despite a tendency towards increased IL-10 and decreased IL-1β, TNFR1, and TNFR2 levels in mTNFΔ/Δ mice. In addition, microglial and leukocyte infiltration, activation state (Iba1, CD11b, CD11c, CD45, and MHCII), lesion size, and functional outcome after moderate SCI were comparable between genotypes. Collectively, our data demonstrate that genetic ablation of solTNF does not significantly modulate postlesion outcome after SCI.