Dupuytren's contracture-associated SNPs increase SFRP4 expression in non-immune cells including fibroblasts to enhance inflammation development.

Dupuytren's contracture (DC) is an inflammatory fibrosis characterized by fibroproliferative disorders of the palmar aponeurosis, for which there is no effective treatment. Although several genome-wide association studies have identified risk alleles associated with DC, the functional linkage between these alleles and the pathogenesis remains elusive. We here focused on two single nucleotide polymorphisms (SNPs) associated with DC, rs16879765 and rs17171229, in secreted frizzled related protein 4 (SFRP4). We investigated the association of SRFP4 with the IL-6 amplifier, which amplifies the production of IL-6, growth factors and chemokines in non-immune cells and aggravates inflammatory diseases via NF-κB enhancement. Knockdown of SFRP4 suppressed activation of the IL-6 amplifier in vitro and in vivo, whereas the overexpression of SFRP4 induced the activation of NF-κB-mediated transcription activity. Mechanistically, SFRP4 induced NF-κB activation by directly binding to molecules of the ubiquitination SFC complex, such as IkBα and ßTrCP, followed by IkBα degradation. Furthermore, SFRP4 expression was significantly increased in fibroblasts derived from DC patients bearing the risk alleles. Consistently, fibroblasts with the risk alleles enhanced activation of the IL-6 amplifier. These findings indicate that the IL-6 amplifier is involved in the pathogenesis of DC, particularly in patients harboring the SFRP4 risk alleles. Therefore, SFRP4 is a potential therapeutic target for various inflammatory diseases and disorders, including DC.

Zoobiquity experiments show the importance of the local MMP9-plasminogen axis in inflammatory bowel diseases in both dogs and patients.

Using a zoobiquity concept, we directly connect animal phenotypes to a human disease mechanism: the reduction of local plasminogen levels caused by matrix metalloproteinase-9 (MMP9) activity is associated with the development of inflammation in the intestines of dogs and patients with inflammatory bowel disease. We first investigated inflammatory colorectal polyps (ICRPs), which are a canine gastrointestinal disease characterized by the presence of idiopathic chronic inflammation, in Miniature Dachshund (MD) and found 31 missense disease-associated SNPs by whole-exome sequencing. We sequenced them in 10 other dog breeds and found five, PLG, TCOF1, TG, COL9A2 and COL4A4, only in MD. We then investigated two rare and breed-specific missense SNPs (T/T SNPs), PLG: c.477G > T and c.478A>T, and found that ICRPs with the T/T SNP risk alleles showed less intact plasminogen and plasmin activity in the lesions compared to ICRPs without the risk alleles but no differences in serum. Moreover, we show that MMP9, which is an NF-κB target, caused the plasminogen reduction and that intestinal epithelial cells expressing plasminogen molecules were co-localized with epithelial cells expressing MMP9 in normal colons with the risk alleles. Importantly, MMP9 expression in patients with ulcerous colitis or Crohn's disease also co-localized with epithelial cells showing enhanced NF-κB activation and less plasminogen expression. Overall, our zoobiquity experiments showed that MMP9 induces the plasminogen reduction in the intestine, contributing to the development of local inflammation and suggesting the local MMP9-plasminogen axis is a therapeutic target in both dogs and patients. Therefore, zoobiquity-type experiments could bring new perspectives for biomarkers and therapeutic targets.

Gateway reflexes, neuronal circuits that regulate the autoreactive T cells in organs having blood barriers.

Gateway reflexes are neural circuits that maintain homeostasis of the immune system. They form gateways for autoreactive T cells to infiltrate the central nervous system in a noradrenaline-dependent manner despite the blood-brain barrier. This mechanism is critical not only for maintaining organ homeostasis but also for inflammatory disease development. Gateway reflexes can be regulated by environmental or artificial stimuli including electrical stimulation, suggesting that the infiltration of immune cells can be controlled by bioelectronic medicine. In this review, we describe the discovery of gateway reflexes and their future directions with special focus on bioelectronic medicine.

Sjögren's syndrome-associated SNPs increase GTF2I expression in salivary gland cells to enhance inflammation development.

Sjögren's syndrome (SS) is an autoimmune disease characterized by inflammation with lymphoid infiltration and destruction of the salivary glands. Although many genome-wide association studies have revealed disease-associated risk alleles, the functions of the majority of these alleles are unclear. Here, we show previously unrecognized roles of GTF2I molecules by using two SS-associated single nucleotide polymorphisms (SNPs), rs73366469 and rs117026326 (GTF2I SNPs). We found that the risk alleles of GTF2I SNPs increased GTF2I expression and enhanced nuclear factor-kappa B (NF-κB) activation in human salivary gland cells via the NF-κB p65 subunit. Indeed, the knockdown of GTF2I suppressed inflammatory responses in mouse endothelial cells and in vivo. Conversely, the over-expression of GTF2I enhanced NF-κB reporter activity depending on its p65-binding N-terminal leucine zipper domain. GTF2I is highly expressed in the human salivary gland cells of SS patients expressing the risk alleles. Consistently, the risk alleles of GTF2I SNPs were strongly associated with activation of the IL-6 amplifier, which is hyperactivation machinery of the NF-κB pathway, and lymphoid infiltration in the salivary glands of SS patients. These results demonstrated that GTF2I expression in salivary glands is increased in the presence of the risk alleles of GTF2I SNPs, resulting in activation of the NF-κB pathway in salivary gland cells. They also suggest that GTF2I could be a new therapeutic target for SS.

IL-6 in inflammation, autoimmunity and cancer.

IL-6 is involved both in immune responses and in inflammation, hematopoiesis, bone metabolism and embryonic development. IL-6 plays roles in chronic inflammation (closely related to chronic inflammatory diseases, autoimmune diseases and cancer) and even in the cytokine storm of corona virus disease 2019 (COVID-19). Acute inflammation during the immune response and wound healing is a well-controlled response, whereas chronic inflammation and the cytokine storm are uncontrolled inflammatory responses. Non-immune and immune cells, cytokines such as IL-1ß, IL-6 and tumor necrosis factor alpha (TNFα) and transcription factors nuclear factor-kappa B (NF-κB) and signal transducer and activator of transcription 3 (STAT3) play central roles in inflammation. Synergistic interactions between NF-κB and STAT3 induce the hyper-activation of NF-κB followed by the production of various inflammatory cytokines. Because IL-6 is an NF-κB target, simultaneous activation of NF-κB and STAT3 in non-immune cells triggers a positive feedback loop of NF-κB activation by the IL-6-STAT3 axis. This positive feedback loop is called the IL-6 amplifier (IL-6 Amp) and is a key player in the local initiation model, which states that local initiators, such as senescence, obesity, stressors, infection, injury and smoking, trigger diseases by promoting interactions between non-immune cells and immune cells. This model counters dogma that holds that autoimmunity and oncogenesis are triggered by the breakdown of tissue-specific immune tolerance and oncogenic mutations, respectively. The IL-6 Amp is activated by a variety of local initiators, demonstrating that the IL-6-STAT3 axis is a critical target for treating diseases.

The gateway reflex: breaking through the blood barriers.

We have been studying inflammatory diseases, with a special focus on IL-6, and discovered two concepts related to inflammation development. One is the gateway reflex, which is induced by the activation of specific neural circuits followed by establishing gateways for autoreactive CD4+ T cells to pass through blood barriers toward the central nervous system (CNS) and retina during tissue-specific inflammatory diseases. We found that the formation of these gateways is dependent on the IL-6 amplifier, which is machinery for enhanced NF-κB activation in endothelial cells at specific sites. We have found five gateway reflexes in total. Here, we introduce the gateway reflex and the IL-6 amplifier.

The gateway reflex regulates tissue-specific autoimmune diseases.

The dynamic interaction and movement of substances and cells between the central nervous system (CNS) and peripheral organs are meticulously controlled by a specialized vascular structure, the blood-brain barrier (BBB). Experimental and clinical research has shown that disruptions in the BBB are characteristic of various neuroinflammatory disorders, including multiple sclerosis. We have been elucidating a mechanism termed the "gateway reflex" that details the entry of immune cells, notably autoreactive T cells, into the CNS at the onset of such diseases. This process is initiated through local neural responses to a range of environmental stimuli, such as gravity, electricity, pain, stress, light, and joint inflammation. These stimuli specifically activate neural pathways to open gateways at targeted blood vessels for blood immune cell entry. The gateway reflex is pivotal in managing tissue-specific inflammatory diseases, and its improper activation is linked to disease progression. In this review, we present a comprehensive examination of the gateway reflex mechanism.

Gateway reflexes are neural circuits that establish the gateway of immune cells to regulate tissue specific inflammation.

INTRODUCTION: Tissue-specific inflammatory diseases are regulated by several mechanisms. The gateway reflex and IL-6 amplifier are two mechanisms involved in diseases that depend on the inflammatory cytokine IL-6. The gateway reflex activates specific neural pathways that cause autoreactive CD4+ T cells to pass through gateways in blood vessels toward specific tissues in tissue-specific inflammatory diseases. These gateways are mediated by the IL-6 amplifier, which describes enhanced NF-κB activation in nonimmune cells including endothelial cells at specific sites. In total, we have reported six gateway reflexes defined by their triggering stimulus: gravity, pain, electric stimulation, stress, light, and joint inflammation. AREAS COVERED: This review summarizes the gateway reflex and IL-6 amplifier for the development of tissue-specific inflammatory diseases. EXPERT OPINION: We expect that the IL-6 amplifier and gateway reflex will lead to novel therapeutic and diagnostic methods for inflammatory diseases, particularly tissue-specific ones.

Gateway reflexes describe novel neuro-immune communications that establish immune cell gateways at specific vessels.

Neuroinflammation is an important biological process induced by complex interactions between immune cells and neuronal cells in the central nervous system (CNS). Recent research on the bidirectional communication between neuronal and immunological systems has provided evidence for how immune and inflammatory processes are regulated by nerve activation. One example is the gateway reflex, in which immune cells bypass the blood brain barrier and infiltrate the CNS to cause neuroinflammation. We have found several modes of the gateway reflex in mouse models, in which gateways for immune cells are established at specific blood vessels in the spinal cords and brain in experimental autoimmune encephalomyelitis and systemic lupus erythematosus models, at retinal blood vessels in an experimental autoimmune uveitis model, and the ankle joints in an inflammatory arthritis model. Several environmental stimulations, including physical and psychological stresses, activate neurological pathways that alter immunological responses via the gateway reflex, thus contributing to the development/suppression of autoimmune diseases. In the manuscript, we describe the discovery of the gateway reflex and recent insights on how they regulate disease development. We hypothesize that artificial manipulation of specific neural pathways can establish and/or close the gateways to control the development of autoimmune diseases.

An inflammatory bowel disease-associated SNP increases local thyroglobulin expression to develop inflammation in miniature dachshunds.

Inflammatory colorectal polyp (ICRP) in miniature dachshunds (MDs) is a chronic inflammatory bowel disease (IBD) characterized by granulomatous inflammation that consists of neutrophil infiltration and goblet cell hyperplasia in the colon. Recently, we identified five MD-associated single-nucleotide polymorphisms (SNPs), namely PLG, TCOF1, TG, COL9A2, and COL4A4, by whole-exome sequencing. Here, we investigated whether TG c.4567C>T (p.R1523W) is associated with the ICRP pathology. We found that the frequency of the T/T SNP risk allele was significantly increased in MDs with ICRP. In vitro experiments showed that TG expression in non-immune cells was increased by inducing the IL-6 amplifier with IL-6 and TNF-α. On the other hand, a deficiency of TG suppressed the IL-6 amplifier. Moreover, recombinant TG treatment enhanced the activation of the IL-6 amplifier, suggesting that TG is both a positive regulator and a target of the IL-6 amplifier. We also found that TG expression together with two NF-κB targets, IL6 and CCL2, was increased in colon samples isolated from MDs with the T/T risk allele compared to those with the C/C non-risk allele, but serum TG was not increased. Cumulatively, these results suggest that the T/T SNP is an expression quantitative trait locus (eQTL) of TG mRNA in the colon, and local TG expression triggered by this SNP increases the risk of ICRP in MDs via the IL-6 amplifier. Therefore, TG c.4567C>T is a diagnostic target for ICRP in MDs, and TG-mediated IL-6 amplifier activation in the colon is a possible therapeutic target for ICRP.

NSAIDs/nitazoxanide/azithromycin repurposed for COVID-19: potential mitigation of the cytokine storm interleukin-6 amplifier via immunomodulatory effects.

INTRODUCTION: Mediators of immunity and inflammation are playing a crucial role in COVID-19 pathogenesis and complications as demonstrated by several genetic and clinical studies. Thus, repurposing of drugs that possess anti-inflammatory and/or immune-modulatory effects for COVID-19 is considered a rational approach. AREAS COVERED: We analyze selected studies that correlated COVID-19 with dysregulated interferon and inflammatory responses while reflecting on our academic and real-life experience using non-steroidal anti-inflammatory drugs, nitazoxanide and azithromycin for management of COVID-19. Moreover, we interpret the results that suggested a potential survival benefit of low-dose aspirin and colchicine when used for COVID-19. EXPERT OPINION: Nitazoxanide/azithromycin combination has been first hypothesized by the author and practiced by him and several researchers to benefit COVID-19 patients due to a potential ability to augment the natural interferon response as well as their positive immunomodulatory effects on several cytokines. Furthermore, NSAIDs, that are unfortunately currently at best of second choice after paracetamol, have been early postulated and clinically practiced by the author to prevent or ameliorate COVID-19 complications and mortality due to their anti-inflammatory and immunomodulatory properties. Finally, we repeat our previous call to adopt our observational study that used these drugs in sufficiently powered double blind randomized clinical trials.