Probes for INS

Peripheral nerve injuries are accompanied by inflammatory reactions, over-activation of which may hinder recovery. Among pro-inflammatory pathways, inflammasomes are one of the most potent, leading to release of active IL-1β. Our aim was to understand how inflammasomes participate in central inflammatory reactions accompanying peripheral nerve injury.After axotomy of the sciatic nerve, priming and activation of the NLRP3 inflammasome was examined in cells of the spinal cord. Regeneration of the nerve was evaluated after coaptation using sciatic functional index measurements and retrograde tracing.In the first 3 days after the injury, elements of the NLRP3 inflammasome were markedly upregulated in the L4-L5 segments of the spinal cord, followed by assembly of the inflammasome and secretion of active IL-1β. Although glial cells are traditionally viewed as initiators of neuroinflammation, in this acute phase of inflammation, inflammasome activation was found exclusively in affected motoneurons of the ventral horn in our model. This process was significantly inhibited by 5-BDBD, a P2X4 receptor inhibitor and MCC950, a potent NLRP3 inhibitor. Although at later time points the NLRP3 protein was upregulated in microglia too, no signs of inflammasome activation were detected in these cells. Inhibition of inflammasome activation in motoneurons in the first days after nerve injury hindered development of microgliosis in the spinal cord. Moreover, P2X4 or inflammasome inhibition in the acute phase significantly enhanced nerve regeneration on both the morphological and the functional levels.Our results indicate that the central reaction initiated by sciatic nerve injury starts with inflammasome activation in motoneurons of the ventral horn, which triggers a complex inflammatory reaction and activation of microglia. Inhibition of neuronal inflammasome activation not only leads to a significant reduction of microgliosis, but has a beneficial effect on the recovery as well.

Central nervous system (CNS)-mediated symptoms, such as fatigue, depression, and hyperalgesia, are common complications among patients with rheumatoid arthritis (RA). However, it remains unclear how the peripheral pathology of RA spreads to the brain. Accumulated evidence showing an association between serum cytokine levels and aberrant CNS function suggests that humoral factors participate in this mechanism. In contrast to the well-known early responses of microglia (CNS-resident immune cells) in the area postrema [AP; a brain region lacking a blood-brain barrier (BBB)] to experimental inflammation, microglial alterations in the AP during chronic inflammation like RA remain unclear. Therefore, to determine whether microglia in the AP can react to persistent autoimmune-arthritis conditions, we analyzed these cells in a mouse model of collagen-induced arthritis (CIA).Microglial number and morphology were analyzed in the AP of CIA and control mice (administered Freund's adjuvant or saline). Immunostaining for ionized calcium-binding adaptor molecule-1 was performed at various disease phases: "pre-onset" [post-immunization day (PID) 21], "establishment" (PID 35), and "chronic" (PID 56 and 84). Quantitative analyses of microglial number and morphology were performed, with principal component analysis used to classify microglia. Interleukin-1β (IL-1β) mRNA expression was analyzed by multiple fluorescent in situ hybridization and real-time polymerase chain reaction. Behavioral changes were assessed by sucrose preference test.Microglia in the AP significantly increased in density and exhibited changes in morphology during the establishment and chronic phases, but not the pre-onset phase. Non-subjective clustering classification of cell morphology (CIA, 1,256 cells; saline, 852 cells) showed that the proportion of highly activated microglia increased in the CIA group during establishment and chronic phases. Moreover, the density of IL-1β-positive microglia, a hallmark of functional activation, was increased in the AP. Sucrose preferences in CIA mice negatively correlated with IL-1β expression in brain regions containing the AP.Our findings demonstrate that microglia in the AP can sustain their activated state during persistent autoimmune arthritis, which suggests that chronic inflammation, such as RA, may affect microglia in brain regions lacking a BBB and have various neural consequences.

Detection of individual cytokines in routine biopsies from patients with inflammatory skin diseases has the potential to personalize diagnosis and treatment selection, but this approach has been limited by technical feasibility. We evaluate whether a chromogen-based RNA in situ hybridization approach can be used to detect druggable cytokines in psoriasis and atopic dermatitis. A series of psoriasis (n = 20) and atopic dermatitis (n = 26) biopsies were stained using RNA in situ hybridization for IL4, IL12B (IL-12/23 p40), IL13, IL17A, IL17F, IL22, IL23A (IL-23 p19), IL31, and TNF (TNF-α). NOS2 and IFNG, canonical psoriasis biomarkers, were also included. All 20 of the psoriasis cases were positive for IL17A, which tended to be the predominant cytokine, although some cases had relatively higher levels of IL12B, IL17F, or IL23A. The majority of cytokine expression in psoriasis was epidermal. A total of 22 of 26 atopic dermatitis cases were positive for IL13, also at varying levels; a subset of cases had significant IL4, IL22, or IL31 expression. Patterns were validated in independent bulk RNA-sequencing and single-cell RNA-sequencing datasets. Overall, RNA in situ hybridization for cytokines appears highly specific with virtually no background staining and may allow for individualized evaluation of treatment-relevant cytokine targets in biopsies from patients with inflammatory skin disorders.

Sarcoidosis is an idiopathic inflammatory disorder that is commonly treated with glucocorticoids. An imprecise understanding of the immunologic changes underlying sarcoidosis has limited therapeutic progress. Here in this open-label trial (NCT03910543), 10 patients with cutaneous sarcoidosis are treated with tofacitinib, a Janus kinase inhibitor. The primary outcome is the change in the cutaneous sarcoidosis activity and morphology instrument (CSAMI) activity score after 6 months of treatment. Secondary outcomes included change in internal organ involvement, molecular parameters, and safety. All patients experience improvement in their skin with 6 patients showing a complete response. Improvement in internal organ involvement is also observed. CD4+ T cell-derived IFN-γ is identified as a central cytokine mediator of macrophage activation in sarcoidosis. Additional type 1 cytokines produced by distinct cell types, including IL-6, IL-12, IL-15 and GM-CSF, also associate with pathogenesis. Suppression of the activity of these cytokines, especially IFN-γ, correlates with clinical improvement. Our results thus show that tofacitinib treatment is associated with improved sarcoidosis symptoms, and predominantly acts by inhibiting type 1 immunity.

Severe cases of SARS-CoV-2 infection are characterized by hypercoagulopathies and systemic endotheliitis of the lung microvasculature. The dynamics of vascular damage, and whether it is a direct consequence of endothelial infection or an indirect consequence of an immune cell-mediated cytokine storm remain unknown. Using a vascularized lung-on-chip model, we find that infection of alveolar epithelial cells leads to limited apical release of virions, consistent with reports of monoculture infection. However, viral RNA and proteins are rapidly detected in underlying endothelial cells, which are themselves refractory to apical infection in monocultures. Although endothelial infection is unproductive, it leads to the formation of cell clusters with low CD31 expression, a progressive loss of barrier integrity and a pro-coagulatory microenvironment. Viral RNA persists in individual cells generating an inflammatory response, which is transient in epithelial cells but persistent in endothelial cells and typified by IL-6 secretion even in the absence of immune cells. Inhibition of IL-6 signalling with tocilizumab reduces but does not prevent loss of barrier integrity. SARS-CoV-2-mediated endothelial cell damage thus occurs independently of cytokine storm.

Microglial-derived inflammation has been linked to a broad range of neurodegenerative and neuropsychiatric conditions, including amyotrophic lateral sclerosis (ALS). Using single-cell RNA sequencing, a class of Disease-Associated Microglia (DAMs) have been characterized in neurodegeneration. However, the DAM phenotype alone is insufficient to explain the functional complexity of microglia, particularly with regard to regulating inflammation that is a hallmark of many neurodegenerative diseases. Here, we identify a subclass of microglia in mouse models of ALS which we term RIPK1-Regulated Inflammatory Microglia (RRIMs). RRIMs show significant up-regulation of classical proinflammatory pathways, including increased levels of Tnf and Il1b RNA and protein. We find that RRIMs are highly regulated by TNFα signaling and that the prevalence of these microglia can be suppressed by inhibiting receptor-interacting protein kinase 1 (RIPK1) activity downstream of the TNF receptor 1. These findings help to elucidate a mechanism by which RIPK1 kinase inhibition has been shown to provide therapeutic benefit in mouse models of ALS and may provide an additional biomarker for analysis in ongoing phase 2 clinical trials of RIPK1 inhibitors in ALS.

Acral dermatoses, including hyperkeratotic palmoplantar eczema (HPE), palmoplantar psoriasis (PP), and mycosis fungoides palmaris et plantaris (MFPP), can be challenging to diagnose clinically and histopathologically. In this setting, cytokine biomarkers may be able to help provide diagnostic clarity. We therefore evaluated interleukin (IL)-17A, interferon gamma (IFN-γ), and IL-13 expression in PP, HPE, and MFPP and compared their expression profiles to non-acral sites. We utilized biopsy specimens from the Yale Dermatopathology database, selecting cases of HPE (n=12), PP (n=8), MFPP (n=8), normal acral skin (n=9), non-acral eczema (n=10), and non-acral psoriasis (n=10) with classic clinical and histopathologic features. IL17A mRNA expression by RNA in situ hybridization differentiated PP (median score 63.1 [IQR 9.4-104.1]) from HPE (0.8 [0-6.0]; P = .003), MFPP (0.6 [0-2.6]; P = .003), and normal acral skin (0 [0-0]; P < .001). Unexpectedly, both PP and HPE demonstrated co-expression of IFNG and IL13 mRNA. In contrast, non-acral psoriasis and eczema demonstrated divergent patterns of IFNG and IL13 mRNA expression. Taken together, we show that IL17A mRNA expression may be a useful biomarker of PP, and we further demonstrate that acral dermatoses exhibit unique immunology compared to non-acral sites, with implications for clinical management.

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.

Elevated levels of cytokines IL-1β and IL-6 are associated with severe COVID-19. Investigating the underlying mechanisms, we find that while primary human airway epithelia (HAE) have functional inflammasomes and support SARS-CoV-2 replication, they are not the source of IL-1β released upon infection. In leukocytes, the SARS-CoV-2 E protein upregulates inflammasome gene transcription via TLR2 to prime, but not activate, inflammasomes. SARS-CoV-2-infected HAE supply a second signal, which includes genomic and mitochondrial DNA, to stimulate leukocyte IL-1β release. Nuclease treatment, STING, and caspase-1 inhibition but not NLRP3 inhibition blocked leukocyte IL-1β release. After release, IL-1β stimulates IL-6 secretion from HAE. Therefore, infection alone does not increase IL-1β secretion by either cell type. Rather, bi-directional interactions between the SARS-CoV-2-infected epithelium and immune bystanders stimulates both IL-1β and IL-6, creating a pro-inflammatory cytokine circuit. Consistent with these observations, patient autopsy lungs show elevated myeloid inflammasome gene signatures in severe COVID-19.