Psoriatic Resolved Skin Epidermal Keratinocytes Retain Disease-Residual Transcriptomic and Epigenomic Profiles.

The disease-residual transcriptomic profile (DRTP) within psoriatic healed/resolved skin and epidermal tissue-resident memory T (TRM) cells have been proposed to be crucial for the recurrence of old lesions. However, it is unclear whether epidermal keratinocytes are involved in disease recurrence. There is increasing evidence regarding the importance of epigenetic mechanisms in the pathogenesis of psoriasis. Nonetheless, the epigenetic changes that contribute to the recurrence of psoriasis remain unknown. The aim of this study was to elucidate the role of keratinocytes in psoriasis relapse. The epigenetic marks 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) were visualized using immunofluorescence staining, and RNA sequencing was performed on paired never-lesional and resolved epidermal and dermal compartments of skin from psoriasis patients. We observed diminished 5-mC and 5-hmC amounts and decreased mRNA expression of the ten-eleven translocation (TET) 3 enzyme in the resolved epidermis. SAMHD1, C10orf99, and AKR1B10: the highly dysregulated genes in resolved epidermis are known to be associated with pathogenesis of psoriasis, and the DRTP was enriched in WNT, TNF, and mTOR signaling pathways. Our results suggest that epigenetic changes detected in epidermal keratinocytes of resolved skin may be responsible for the DRTP in the same regions. Thus, the DRTP of keratinocytes may contribute to site-specific local relapse.

Anti-inflammatory effect of chymotrypsin to autoimmune response against CNS is dose-dependent.

Multiple sclerosis is an inflammatory autoimmune disease of central nervous system (CNS) in which inflammatory cells release pro-inflammatory cytokines, proteases, and other toxic mediators. Proteases are involved in many aspects of inflammatory process. There are many reports regarding the effect of proteases on inflammation. Chymotrypsin is a serine protease with anti-inflammatory effect. We investigated chymotrypsin effect on experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis. Intra-CSF injection with 0.1 mg/ml, 0.2 mg/ml chymotrypsin, or saline was done on day 7 after EAE induction. Our study demonstrated that 0.1 mg/ml chymotrypsin treatment did not decrease clinical signs, but 0.2 mg/ml chymotrypsin ameliorated clinical signs and manipulated immune response in both brain and spinal cord. Administration of 0.1 mg/ml or 0.2 mg/ml chymotrypsin led to decreased IL-17 along with increased IL-4 and FoxP3 in 0.2 mg/ml chymotrypsin-treated animals. Presumably, chymotrypsin acts in a dose-dependent manner and concentrations of chymotrypsin more than 0.2 mg/ml may have more beneficial effect.

Three-dimensional bioprinting of functional ß-islet-like constructs.

256Diabetes is an autoimmune disease that ensues when the pancreas does not deliver adequate insulin or when the body cannot react to the existing insulin. Type 1 diabetes is an autoimmune disease defined by continuous high blood sugar levels and insulin deficiency due to ß-cell destruction in the islets of Langerhans (pancreatic islets). Long-term complications, such as vascular degeneration, blindness, and renal failure, result from periodic glucose-level fluctuations following exogenous insulin therapy. Nevertheless, the shortage of organ donors and the lifelong dependency on immunosuppressive drugs limit the transplantation of the entire pancreas or pancreas islet, which is the therapy for this disease. Although encapsulating pancreatic islets using multiple hydrogels creates a semi-privileged environment to prevent immune rejection, hypoxia that occurs in the core of the capsules is the main hindrance that should be solved. Bioprinting technology is an innovative process in advanced tissue engineering that allows the arranging of a wide array of cell types, biomaterials, and bioactive factors as a bioink to simulate the native tissue environment for fabricating clinically applicable bioartificial pancreatic islet tissue. Multipotent stem cells have the potential to be a possible solution for donor scarcity and can be a reliable source for generating autograft and allograft functional ß-cells or even pancreatic islet-like tissue. The use of supporting cells, such as endothelial cells, regulatory T cells, and mesenchymal stem cells, in the bioprinting of pancreatic islet-like construct could enhance vasculogenesis and regulate immune activity. Moreover, scaffolds bioprinted using biomaterials that can release oxygen postprinting or enhance angiogenesis could increase the function of ß-cells and the survival of pancreatic islets, which could represent a promising avenue.

Abnormal basement membrane results in increased keratinocyte-derived periostin expression in psoriasis similar to wound healing.

The psoriatic skin resembles wound healing, and it shows abnormalities at the basement membrane (BM), also in the non-lesional skin. Fibroblast-derived dermal periostin has well-known functions in wound healing and Th2-mediated diseases, such as atopic dermatitis. Here we show that serum periostin level was elevated in psoriatic patients, remarkably in the systemically treated ones. Obvious periostin positivity was detected in basal keratinocytes of the non-lesional, lesional, and previously-lesional psoriatic vs. healthy skin. Ex vivo skin models were generated to examine how different skin injuries affect periostin expression during wound healing. Our newly developed cultured salt-split model demonstrated that BM-injury induced periostin expression in basal keratinocytes, and periostin levels in the supernatant were also increased upon healing. In wound healing models, ß1-integrin expression was similarly induced. ß1-integrin blocking caused reduced periostin expression in in vitro scratch assay, indicating that ß1-integrin can mediate periostin production. In contrast to atopic dermatitis, psoriatic basal keratinocytes are in an activated state and show a stable wound healing-like phenotype with the overexpression of periostin. This abnormal BM-induced wound healing as a potential compensatory mechanism can be initiated already in the non-lesional skin present in the lesion and keratinocytes can remain activated in the healed skin.

Unraveling Transcriptome Profile, Epigenetic Dynamics, and Morphological Changes in Psoriasis-like Keratinocytes: "Insights into Similarity with Psoriatic Lesional Epidermis".

Keratinocytes are one of the primary cells affected by psoriasis inflammation. Our study aimed to delve deeper into their morphology, transcriptome, and epigenome changes in response to psoriasis-like inflammation. We created a novel cytokine mixture to mimic mild and severe psoriasis-like inflammatory conditions in cultured keratinocytes. Upon induction of inflammation, we observed that the keratinocytes exhibited a mesenchymal-like phenotype, further confirmed by increased VIM mRNA expression and results obtained from confocal microscopy. We performed RNA sequencing to achieve a more global view, revealing 858 and 6987 DEGs in mildly and severely inflamed keratinocytes, respectively. Surprisingly, we found that the transcriptome of mildly inflamed keratinocytes more closely mimicked that of the psoriatic epidermis transcriptome than the severely inflamed keratinocytes. Genes involved in the IL-17 pathway were a major contributor to the similarities of the transcriptomes between mildly inflamed KCs and psoriatic epidermis. Mild and severe inflammation led to the gene regulation of epigenetic modifiers such as HATs, HDACs, DNMTs, and TETs. Immunofluorescence staining revealed distinct 5-hmC patterns in inflamed versus control keratinocytes, and consistently low 5-mC intensity in both groups. However, the global DNA methylation assay detected a tendency of decreased 5-mC levels in inflamed keratinocytes versus controls. This study emphasizes how inflammation severity affects the transcriptomic similarity of keratinocytes to psoriatic epidermis and proves dynamic epigenetic regulation and adaptive morphological changes in inflamed keratinocytes.

Kinetics of T cell response in the testes and CNS during experimental autoimmune encephalomyelitis: Simultaneous blood-brain and -testis barrier permeability?

OBJECTIVES: Multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), are regarded as autoimmune diseases of the central nervous system (CNS). The CNS, testes, and eyes are immune privileged sites. It was initially presumed that ocular involvement in EAE and infertility in MS are neural-mediated. However, inflammatory molecules have been detected in the eyes of animals affected by EAE. It prompted us to investigate if the testes may also be targeted by immune response during EAE. MATERIALS AND METHODS: kinetics of T cell response was investigated in the CNS and testes in EAE at different clinical scores. IFN-γ, IL-4, IL-17, and FoxP3 mRNA expressions were considered as representatives of Th1, Th2, Th17, and Treg, respectively. RESULTS: In CNS, IL-17 and IFN-γ were initially up-regulated and attenuated at the late phase of the disease. IL-4 and FoxP3 were markedly down-regulated, but IL-4 was then up-regulated at the late phase of the disease. In the testes, IFN-γ and IL-17 were diminished but increased at the late phase of the disease. FoxP3 was gradually increased from the initial step to the peak of the disease. IL-17/ IFN-γ showed a similar pattern between the CNS and testes. However, FoxP3 and IL-4 expression appeared to have different timing patterns in the CNS and testes. CONCLUSION: Given the permeability in blood-retina/brain/CSF barrier by complete Freund's adjuvant, the pattern of T cells may be changed in the testes during EAE as a consequence of the blood-testis barrier permeability. More research is required to explore the connection between immune privileged organs.

Neuroimmunomodulation by allogeneic seminal vesicle fluid in CNS is sex-independent.

OBJECTIVES: Thymus-arisen FoxP3 regulatory T cells (Tregs) are one of the most important immunoregulatory mechanisms in the central nervous system (CNS) and pregnancy. Multiple sclerosis (MS) is an inflammatory disease of CNS associated with a reduced frequency and/or function of Tregs. Previous works have shown that seminal vesicle fluid affects female immune system and causes expansion of Tregs pool in the female reproductive tissue upon mating. Accordingly, it has been demonstrated that intra-CSF administration of seminal vesicle fluid from Wistar rats can ameliorate clinical sign of a female Lewis rat model of experimental autoimmune encephalomyelitis (EAE), the animal model of MS. The results indicated an up-regulation of FoxP3 expression in the brain and spinal cord. However, there are sex-based differences in the CNS structure & composition, sex hormones influence immune system, and gender-based differences affect treatment response. Therefore, we decided to find out if anti-inflammatory effect of seminal vesicle fluid is sex-dependent or -independent. METHODS: EAE was induced in male Lewis rats using guinea pig spinal cord and complete Freund's adjuvant. Intra-CSF injection was done on day 7 after EAE induction and the animals were followed-up until on day 14 after EAE induction when sacrificed. Then, brain and spinal cord of the animals were isolated, total RNA was extracted, and expression of mRNA for IFN-γ, IL-4, IL-17, and FoxP3 was determined using real-time PCR where ß-actin was used as reference gene. RESULT: demonstrated that intra-CSF administration of seminal vesicle fluid from male Wistar rats ameliorated EAE in male Lewis rats and increased FoxP3 in the brain and spinal cord. CONCLUSION: This study suggests that seminal vesicle fluid from Wistar rats has anti-inflammatory effect on Lewis rats in a sex-independent manner. In addition, seminal vesicle fluid from Lewis rats had not beneficial effect on EAE in male Lewis rats. This is consistent with Tregs increase in allogeneic mating. More research is required to find out the immunologic aspect of allogeneic versus syngeneic administration of seminal vesicle fluid.

Immunomodulatory Effect of Chymotrypsin in CNS Is Sex-independent: Evidence of Anti-inflammatory Role for IL-17 in EAE.

Multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), are inflammatory autoimmune diseases of the central nervous system. Chymotrypsin is a serine protease with immunomodulatory effect in the peripheral organs. We previously demonstrated the immunomodulatory effect of chymotrypsin in ameliorating the EAE in female Lewis rats. However, there are sex-based differences in the immune system, drug activity, and CNS structure and composition. In addition, female gender is a better prognostic indicator of MS and males are more severely affected by EAE than females. Consequently, gender may have an important impact on therapeutic effect. Therefore, in this study we investigated the anti-inflammatory effect of chymotrypsin in male Lewis rat model of EAE. The disease was induced in male Lewis rats and the animals were evaluated for weight loss and clinical signs for 14 days. Intra-CSF injection of chymotrypsin was done on day 7 and expression of mRNA for IFN-γ, IL-4, IL-17, and FoxP3 in brain, spinal cord and deep cervical lymph node were determined using a two-step real-time PCR. Administration of 0.2mg/ml chymotrypsin ameliorated the disease by decreasing IFN-γ and increasing expression of IL-4 and IL-17 at the inflammatory foci. This is consistent with anti-inflammatory effect of IL-4 and IL-17 at high concentrations. We conclude that Immunomodulatory affect of chymotrypsin in CNS is sex-independent. Our result also provides more evidence on the anti-inflammatory role of IL-17. However more research is needed to elucidate the underlying immunomodulatory role of chymotrypsin and how to increase its beneficial effect by modification of dosage and/or regimen of administration.

Liver Damage and Mortality in a Male Lewis Rat of Experimental Autoimmune Encephalomyelitis.

BACKGROUND AND OBJECTIVES: Multiple sclerosis is an inflammatory disease of the central nervous system. This is due to migration of peripherally activated lymphocytes to central nervous system leading to inflammatory lesions. However, liver has an anti-inflammatory microenvironment. Myelin expression in the liver of transgenic mice suppresses inflammatory lesions within central nervous system. Considering the notion that the inflammatory events originate from periphery, we investigated if the liver was affected in an animal model for multiple sclerosis. METHODS: Experimental autoimmune encephalomyelitis was induced in male Lewis rats using guinea pig spinal cord and complete Freund's adjuvant. Weight, clinical score, and survival rate were evaluated for 14 days post immunization. Liver sections were taken and stained with Hematoxylin and Eosin and examined with an Olympus microscope. RESULTS: Mortality was accompanied by liver damage. Sinusoidal congestion, pycnotic nuclei within hepatocytes, hepatocyte necrosis, and severe widespread congestion along with fat accumulation within hepatocytes (fatty degeneration) were observed in liver tissue sections. CONCLUSION: Liver damage occurs in experimental autoimmune encephalomyelitis. The perpetuation of self antigen leading to continuous migration of extrahepatically activated T cells makes an inflammatory milieu in the liver. It follows migration and development of more inflammatory cells and may paralyses tolerance inducing mechanisms. Apart from central nervous system lesion, liver injury may act as synergistic factor for debilitation and mortality.