Probes for INS

An early inflammatory insult is the most recognized risk factor associated with neurodevelopmental psychiatric disorders, even more so than genetic variants. Notably, complement component 4 (C4), a molecule involved in inflammatory responses, has been strongly associated with schizophrenia (SZ) and its role in other neurodevelopmental disorders, such as autism (ASD), is an area of active investigation. However, while C4 in SZ has been implicated in the context of synaptic pruning, little is known about its neuroinflammatory role. The subventricular zone (SVZ) is a region heavily involved in neurodevelopment and neuroimmune interactions through the lifespan; thus, it is a region wherein C4 may play a vital role in disease pathology. Using in situ hybridization with radioactive riboprobes and RNAscope, we identified robust astrocytic expression of C4 in the SVZ and in the septum pellucidum. C4 was also expressed in ependyma, neurons, and Ki67+ progenitor cells. Examination of mRNA levels showed elevated C4 in both ASD and SZ, with higher expression in SZ compared to controls. Targeted transcriptomic analysis of inflammatory pathways revealed a strong association of complement system genes with SZ, and to a lesser extent, ASD, as well as generalized immune dysregulation without a strong association with known infectious pathways. Analysis of differentially expressed genes (DEGs) showed that ASD DEGs were enriched in adaptive immune system functions such as Th cell differentiation, while SZ DEGs were enriched in innate immune system functions, including NF-κB and toll like receptor signaling. Moreover, the number of Ki67+ cells was significantly higher in ASD compared to SZ and controls. Taken together, these results support a role for C4 into inflammatory-neuroimmune dysregulation observed in SZ and ASD pathology.

Renal interstitial fibrosis is characterized by the development of myofibroblasts, originating from resident renal and immigrating cells. Myofibroblast formation and extracellular matrix production during kidney damage are triggered by various factors. Among these, endothelins have been discussed as potential modulators of renal fibrosis. Utilizing mouse models of adenine nephropathy (AN) and unilateral ureter occlusion (UUO), this study aimed to investigate the contribution of endothelin signaling in stromal mesenchymal resident renal interstitial cells. We found in controls that adenine feeding and UUO caused marked upregulations of endothelin-1 (ET-1) gene expression in endothelial and in tubular cells and a strong upregulation of ETA-receptor (ETA-R) gene expression in interstitial and mesangial cells, while the gene expression of ETB-receptor (ETB-R) did not change. Conditional deletion of ETA-R and ETB-R gene expression in the FoxD1 stromal cell compartment which includes interstitial cells significantly reduced renal ETA-R gene expression and moderately lowered renal ETB-R gene expression. ET receptor (ET-R) deletion exerted no apparent effects on kidney development nor on kidney function. Adenine feeding and UUO led to similar increases in profibrotic and proinflammatory gene expression in control as well as in ETAflflETBflfl FoxD1Cre+ mice (ET-Ko). In summary, our findings suggest that adenine feeding and UUO activate endothelin signaling in interstitial cells which is due to upregulated ETA-R expression and enhanced renal ET-1 production Our data also suggest that the activation of endothelin signaling in interstitial cells has less impact for the development of experimentally induced fibrosis.

Astrocytic activation might play a significant role in the central sensitization of chronic migraine (CM). However, the temporal characteristics of the astrocytic activation in the trigeminal nucleus caudalis (TNC) and the molecular mechanism under the process remain not fully understood. Therefore, this study aims to investigate the duration and levels change of astrocytic activation and to explore the correlation between astrocytic activation and the levels change of cytokines release.We used a mice model induced by recurrent dural infusion of inflammatory soup (IS). The variation with time of IS-induced mechanical thresholds in the periorbital and hind paw plantar regions were evaluated using the von Frey filaments test. We detected the expression profile of glial fibrillary acidic protein (GFAP) in the TNC through immunofluorescence staining and western blot assay. We also investigated the variation with time of the transcriptional levels of GFAP and ionized calcium binding adapter molecule 1 (Iba1) through RNAscope in situ hybridization analysis. Then, we detected the variation with time of cytokines levels in the TNC tissue extraction and serum, including c-c motif chemokine ligand 2 (CCL2), c-c motif chemokine ligand 5 (CCL5), c-c motif chemokine ligand 7 (CCL7), c-c motif chemokine ligand 12 (CCL12), c-x-c motif chemokine ligand 1 (CXCL1), c-x-c motif chemokine ligand 13 (CXCL13), interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), macrophage colony-stimulating factor (M-CSF), interleukin 1beta (IL-1β), interleukin 6 (IL-6), interleukin 10 (IL-10), interleukin 17A (IL-17A).Recurrent IS infusion resulted in cutaneous allodynia in both the periorbital region and hind paw plantar, ranging from 5 d (after the second IS infusion) to 47 d (28 d after the last infusion) and 5 d to 26 d (7 d after the last infusion), respectively. The protein levels of GFAP and messenger ribonucleic acid (mRNA) levels of GFAP and Iba1 significantly increased and sustained from 20 d to 47 d (1 d to 28 d after the last infusion), which was associated with the temporal characteristics of astrocytic activation in the TNC. The CCL7 levels in the TNC decreased from 20 d to 47 d. But the CCL7 levels in serum only decreased on 20 d (1 d after the last infusion). The CCL12 levels in the TNC decreased on 22 d (3 d after the last infusion) and 33 d (14 d after the last infusion). In serum, the CCL12 levels only decreased on 22 d. The IL-10 levels in the TNC increased on 20 d.Our results indicate that the astrocytic activation generated and sustained in the IS-induced mice model from 1 d to 28 d after the last infusion and may contribute to the pathology through modulating CCL7, CCL12, and IL-10 release.

The sequence of pathological events in feline hypertrophic cardiomyopathy (fHCM) is still largely unknown, although we know that fHCM is characterized by interstitial remodeling in a macrophage-driven pro-inflammatory environment and that myocardial ischemia might contribute to its progression. This study aimed to gain further insights into the structural changes associated with interstitial remodeling in fHCM with special focus on the myocardial microvasculature and the phenotype of the interstitial cells. Twenty-eight hearts (16 hearts with fHCM and 12 without cardiac disease) were evaluated in the current study, with immunohistochemistry, RNA-in situ hybridization, and transmission electron microscopy. Morphometrical evaluations revealed a statistically significant lower microvascular density in fHCM. This was associated with structural alterations in capillaries that go along with a widening of the interstitium due to the accumulation of edema fluid, collagen fibers, and mononuclear cells that also proliferated locally. The interstitial cells were mainly of fibroblastic or vascular phenotype, with a substantial contribution of predominantly resident macrophages. A large proportion expressed CD34 mRNA, which suggests a progenitor cell potential. Our results indicate that microvascular alterations are key events in the pathogenesis of fHCM and that myocardial interstitial cell populations with CD34+ phenotype play a role in the pathogenesis of the disease.

Sprouty1 (Spry1) regulates the differentiation of vascular smooth muscle cells (VSMC), and our aim was to determine its role in atherogenesis. A significant proportion of cells within atherosclerotic lesions are derived from migration and pathological adaptation of medial VSMC.We used global Spry1 null mouse, and Myh11-CreERT2, ROSA26-STOPfl/fl-tdTomato-Spry1fl/fl mice to allow for lineage tracing and conditional Spry1 deletion in VSMC. Atherosclerosis was induced by injection of a mutant form of mPCSK9D377Y-AAV followed by Western diet. Human aortic VSMC (hVSMC) with shRNA targeting of Spry1 were also analyzed.Global loss of Spry1 increased inflammatory markers ICAM1 and Cox2 in VSMC. Conditional deletion of Spry1 in VSMC had no effect on early lesion development, despite increased Sca1high cells. After 26 weeks of Western diet, mice with VSMC deletion of Spry1 had increased plaque burden, with reduced collagen content and smooth muscle alpha actin (SMA) in the fibrous cap. Lineage tracing via tdTomato marking Cre-recombined cells indicated that VSMC with loss of Spry1 had decreased migration into the lesion, noted by decreased proportions of tdTomato+ and tdTomato+/SMA + cells. Loss-of-function of Spry1 in hVSMC increased mesenchymal and activation markers, including KLF4, PDGFRb, ICAM1, and Cox2. Loss of Spry1 enhanced the effects of PDGFBB and TNFa on hVSMC.Loss of Spry1 in VSMC aggravated plaque formation at later stages, and increased markers of instability. Our results indicate that Spry1 suppresses the mesenchymal and inflammatory phenotype of VSMC, and its expression in VSMC is protective against chronic atherosclerotic disease.

Spinal astrocytes contribute to chronic itch via sensitization of itch-specific neurons expressing gastrin-releasing peptide receptor (GRPR). However, whether microglia-neuron interactions contribute to itch remains unclear. In this study, we aimed to explore how microglia interact with GRPR+ neurons and promote chronic itch.RNA sequencing, quantitative real-time PCR, western blot, immunohistochemistry, RNAscope ISH, pharmacologic and genetic approaches were performed to examine the roles of spinal NLRP3 (The NOD-like receptor family, pyrin-containing domain 3) inflammasome activation and IL-1β-IL1R1 signaling in chronic itch. Grpr-eGFP and Grpr KO mice were used to investigate microglia-GRPR+ neuron interactions.We observed NLRP3 inflammasome activation and IL-1β production in spinal microglia under chronic itch conditions. Blockade of microglial activation and the NLRP3/caspase-1/IL-1β axis attenuated chronic itch and neuronal activation. Type 1 IL-1 receptor (IL-1R1) was expressed in GRPR+ neurons, which are essential for the development of chronic itch. Our studies also find that IL-1β+ microglia are localized in close proximity to GRPR+ neurons. Consistently, intrathecal injection of IL1R1 antagonist or exogenous IL-1β indicate that the IL-1β-IL-1R1 signaling pathway enhanced the activation of GRPR+ neurons. Furthermore, our results demonstrate that the microglial NLRP3/caspase-1/IL-1β axis contributes to several different chronic itches triggered by small molecules and protein allergens from the environment and drugs.Our findings reveal a previously unknown mechanism in which microglia enhances the activation of GRPR+ neurons through the NLRP3/caspase-1/IL-1β/IL1R1 axis. These results will provide new insights into the pathophysiology of pruritus and novel therapeutic strategies for patients with chronic itch.

Ataxia-telangiectasia (A-T) is a genetic disorder caused by the lack of functional ATM kinase. A-T is characterized by chronic inflammation, neurodegeneration and premature ageing features that are associated with increased genome instability, nuclear shape alterations, micronuclei accumulation, neuronal defects and premature entry into cellular senescence. The causal relationship between the detrimental inflammatory signature and the neurological deficiencies of A-T remains elusive. Here, we utilize human pluripotent stem cell-derived cortical brain organoids to study A-T neuropathology. Mechanistically, we show that the cGAS-STING pathway is required for the recognition of micronuclei and induction of a senescence-associated secretory phenotype (SASP) in A-T olfactory neurosphere-derived cells and brain organoids. We further demonstrate that cGAS and STING inhibition effectively suppresses self-DNA-triggered SASP expression in A-T brain organoids, inhibits astrocyte senescence and neurodegeneration, and ameliorates A-T brain organoid neuropathology. Our study thus reveals that increased cGAS and STING activity is an important contributor to chronic inflammation and premature senescence in the central nervous system of A-T and constitutes a novel therapeutic target for treating neuropathology in A-T patients.

Astrocytes are vital support cells that ensure proper brain function. In brain disease, astrocytes reprogram into a reactive state that alters many of their cellular roles. A long-standing question in the field is whether downregulation of reactive astrocyte (RA) markers during resolution of inflammation is because these astrocytes revert back to a non-reactive state or die and are replaced. This has proven difficult to answer mainly because existing genetic tools cannot distinguish between healthy versus RAs. Here we describe the generation of an inducible genetic tool that can be used to specifically target and label a subset of RAs. Longitudinal analysis of an acute inflammation model using this tool revealed that the previously observed downregulation of RA markers after inflammation is likely due to changes in gene expression and not because of cell death. Our findings suggest that cellular changes associated with astrogliosis after acute inflammation are largely reversible.