Interleukin-22: immunobiology and pathology.

Interleukin-22 (IL-22) is a recently described IL-10 family cytokine that is produced by T helper (Th) 17 cells, γδ T cells, NKT cells, and newly described innate lymphoid cells (ILCs). Knowledge of IL-22 biology has evolved rapidly since its discovery in 2000, and a role for IL-22 has been identified in numerous tissues, including the intestines, lung, liver, kidney, thymus, pancreas, and skin. IL-22 primarily targets nonhematopoietic epithelial and stromal cells, where it can promote proliferation and play a role in tissue regeneration. In addition, IL-22 regulates host defense at barrier surfaces. However, IL-22 has also been linked to several conditions involving inflammatory tissue pathology. In this review, we assess the current understanding of this cytokine, including its physiologic and pathologic effects on epithelial cell function.

Regulation and functions of the IL-10 family of cytokines in inflammation and disease.

The IL-10 family of cytokines consists of nine members: IL-10, IL-19, IL-20, IL-22, IL-24, IL-26, and the more distantly related IL-28A, IL-28B, and IL-29. Evolutionarily, IL-10 family cytokines emerged before the adaptive immune response. These cytokines elicit diverse host defense mechanisms, especially from epithelial cells, during various infections. IL-10 family cytokines are essential for maintaining the integrity and homeostasis of tissue epithelial layers. Members of this family can promote innate immune responses from tissue epithelia to limit the damage caused by viral and bacterial infections. These cytokines can also facilitate the tissue-healing process in injuries caused by infection or inflammation. Finally, IL-10 itself can repress proinflammatory responses and limit unnecessary tissue disruptions caused by inflammation. Thus, IL-10 family cytokines have indispensable functions in many infectious and inflammatory diseases.

IL-12 family cytokines: immunological playmakers.

The interleukin 12 (IL-12) family is unique in having the only heterodimeric cytokines, including IL-12, IL-23, IL-27 and IL-35. This feature endows these cytokines with a unique set of connections and functional interactions not shared by other cytokine families. Despite sharing many structural features and molecular partners, cytokines of the IL-12 family mediate surprisingly diverse functional effects. Here we discuss the unique and unusual structural and functional characteristics of this cytokine family. We outline how cells might interpret seemingly similar cytokine signals to give rise to the diverse functional outcomes that characterize this cytokine family. We also discuss the therapeutic implications of this complexity.

Elucidation of the Reversible Self-Association Interface of a Diabody-Interleukin Fusion Protein Using Hydrogen-Exchange Mass Spectrometry and In Silico Modeling.

Reversible self-association (RSA) of therapeutic proteins presents major challenges in the development of high-concentration formulations, especially those intended for subcutaneous administration. Understanding self-association mechanisms is therefore critical to the design and selection of candidates with acceptable developability to advance to clinical trials. The combination of experiments and in silico modeling presents a powerful tool to elucidate the interface of self-association. RSA of monoclonal antibodies has been studied extensively under different solution conditions and have been shown to involve interactions for both the antigen-binding fragment and the crystallizable fragment. Novel modalities such as bispecific antibodies, antigen-binding fragments, single-chain-variable fragments, and diabodies constitute a fast-growing class of antibody-based therapeutics that have unique physiochemical properties compared to monoclonal antibodies. In this study, the RSA interface of a diabody-interleukin 22 fusion protein (FP-1) was studied using hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) in combination with in silico modeling. Taken together, the results show that a complex solution behavior underlies the self-association of FP-1 and that the interface thereof can be attributed to a specific segment in the variable light chain of the diabody. These findings also demonstrate that the combination of HDX-MS with in silico modeling is a powerful tool to guide the design and candidate selection of novel biotherapeutic modalities.

Cut-off value for interleukin-34 as an additional potential inflammatory biomarker for estimation of slow coronary flow risk.

BACKGROUND: Inflammatory markers may provide insights into the underlying mechanisms of slow coronary flow (SCF), including subclinical atherosclerosis and endothelial dysfunction. Interleukin-34 (IL-34), known for its role in immuno-inflammatory diseases, might hold significance in SCF. We aimed to explore the potential association between IL-34 and SCF in patients undergoing diagnostic elective coronary angiography. METHODS: This observational, cross-sectional study enrolled 256 participants: 124 with SCF and 132 with normal coronary flow (NCF). All participants had undergone outpatient coronary angiography for suspected coronary artery disease. SCF assessment employed the TIMI frame count (TFC) for quantifying coronary flow rate. RESULTS: SCF patients exhibited significantly elevated TFC in all three major coronary arteries compared to controls (p < 0.05). IL-34 displayed a noteworthy positive correlation with average TFC [for all participants: r = 0.514, p < 0.001; for SCF patients: r = 0.526, p < 0.001; for normal controls: r = -0.288, p > 0.05]. Similarly, high-sensitivity C-reactive protein (hsCRP) showed a significant and positive relationship with average TFC [for all participants: r = 0.504, p < 0.001; for SCF patients: r = 0.558, p < 0.001; for normal controls: r = -0.148, p > 0.05]. SCF patients presented coronary arteries of larger size compared to controls. CONCLUSION: Mean coronary diameter and IL-34 emerged as independent predictors of SCF. Additionally, hsCRP, mean coronary diameter, and IL-34 exhibited a positive correlation with mean TFC values. IL-34 appears to be a more effective indicator than hsCRP in SCF patients.

A comprehensive review of IL-26 to pave a new way for a profound understanding of the pathobiology of cancer, inflammatory diseases and infections.

Cytokines are considered vital mediators of the immune system. Down- or upregulation of these mediators is linked to several inflammatory and pathologic situations. IL-26 is referred to as an identified member of the IL-10 family and IL-20 subfamily. Due to having a unique cationic structure, IL-26 exerts diverse functions in several diseases. Since IL-26 is mainly secreted from Th17, it is primarily considered a pro-inflammatory cytokine. Upon binding to its receptor complex (IL-10R1/IL-20R2), IL-26 activates multiple signalling mediators, especially STAT1/STAT3. In cancer, IL-26 induces IL-22-producing cells, which consequently decrease cytotoxic T-cell functions and promote tumour growth through activating anti-apoptotic proteins. In hypersensitivity conditions such as rheumatoid arthritis, multiple sclerosis, psoriasis and allergic disease, this cytokine functions primarily as the disease-promoting mediator and might be considered a biomarker for disease prognosis. Although IL-26 exerts antimicrobial function in infections such as hepatitis, tuberculosis and leprosy, it has also been shown that IL-26 might be involved in the pathogenesis and exacerbation of sepsis. Besides, the involvement of IL-26 has been confirmed in other conditions, including graft-versus-host disease and chronic obstructive pulmonary disease. Therefore, due to the multifarious function of this cytokine, it is proposed that the underlying mechanism regarding IL-26 function should be elucidated. Collectively, it is hoped that the examination of IL-26 in several contexts might be promising in predicting disease prognosis and might introduce novel approaches in the treatment of various diseases.

Evaluation of interleukins 37 and 38 and vitamin D status in the serum of women with Graves' disease.

BACKGROUND: Graves' disease (GD) is an autoimmune thyroid disorder and recent studies have proposed a role for interleukin (IL)-37, IL-38, and vitamin D (VitD) in the pathophysiology of disease. Therefore, this study investigated the expression of IL-37, IL-38, and VitD in the serum of GD patients and correlations of their levels with some demographic and clinical characteristics. METHODS: Serum IL-37, IL-38, and VitD levels were evaluated in 90 women with GD and 93 control women using enzyme-linked immunosorbent assay kits. Depending on therapy, six patients were newly diagnosed (ND; untreated), and 50 patients were receiving only carbimazole (CMZ), while 34 patients were also on CMZ but also received one (31 patients), two (one patient), or three (two patients) doses of radioactive iodine (RAI). RESULTS: IL-37 levels were significantly higher in GD patients than in controls, while IL-38 and VitD levels were significantly decreased. As indicated by the area under the curve (AUC), receiver operating characteristic curve analysis demonstrated the potential of IL-37, IL-38, and VitD as biomarkers to distinguish GD patients from controls (AUC = 0.953, 0.959, and 0.793, respectively). Multinomial logistic regression analysis showed that altered levels of IL-37, IL-38, and VitD were most likely associated with the pathogenesis of GD. IL-37 was negatively correlated with IL-38 and VitD, while IL-38 and VitD were positively correlated. CONCLUSION: Serum Il-37 levels were upregulated in women with GD, while IL-38 and VitD levels showed downregulated levels. The latter two were positively correlated while they showed a negative correlation with IL-37.

Interleukin-22: a potential therapeutic target in atherosclerosis.

BACKGROUND: Atherosclerosis is recognized as a chronic immuno-inflammatory disease that is characterized by the accumulation of immune cells and lipids in the vascular wall. In this review, we focus on the latest advance regarding the regulation and signaling pathways of IL-22 and highlight its impacts on atherosclerosis. MAIN BODY: IL-22, an important member of the IL-10 family of cytokines, is released by cells of the adaptive and innate immune system and plays a key role in the development of inflammatory diseases. The binding of IL-22 to its receptor complex can trigger a diverse array of downstream signaling pathways, in particular the JAK/STAT, to induce the expression of chemokines and proinflammatory cytokines. Recently, numerous studies suggest that IL-22 is involved in the pathogenesis of atherosclerosis by regulation of VSMC proliferation and migration, angiogenesis, inflammatory response, hypertension, and cholesterol metabolism. CONCLUSION: IL-22 promotes the development of atherosclerosis by multiple mechanisms, which may be a promising therapeutic target in the pathogenesis of atherosclerosis.

A method for making alignments of related protein sequences that share very little similarity; shark interleukin 2 as an example.

An optimized alignment of related protein sequences helps to see their important shared features and to deduce their phylogenetic relationships. At low levels of sequence similarity, there are no suitable computer programs for making the best possible alignment. This review summarizes some guidelines for how in such instances, nevertheless, insightful alignments can be made. The method involves, basically, the understanding of molecular family features at both the protein and intron-exon level, and the collection of many related sequences so that gradual differences may be observed. The method is exemplified by identifying and aligning interleukin 2 (IL-2) and related sequences in Elasmobranchii (sharks/rays) and coelacanth, as other authors have expressed difficulty with their identification. From the point of general immunology, it is interesting that the unusual long "leader" sequence of IL-15, already known in other species, is even more impressively conserved in cartilaginous fish. Furthermore, sequence comparisons suggest that IL-2 in cartilaginous fish has lost its ability to bind an IL-2Rα/15Rα receptor chain, which would prohibit the existence of a mechanism for regulatory T cell regulation identical to mammals.

Functional characterization of an interleukin 20 like homologue in grass carp Ctenopharyngodon idella.

IL-20 is a pleiotropic cytokine that belongs to the IL-10 family and plays an important biological role in tissue homeostasis and regulation of host immune defenses. IL-20 homologues have recently been discovered in fish, but their functions have not been studied. In this study, an IL-20 like (IL-20L) cytokine was cloned in grass carp (Ctenopharyngodon idella) and its bioactivities were investigated. Expression analysis showed that the CiIL-20L gene was constitutively expressed in tissues with the highest expression detected in the head kidney. It was upregulated in the head kidney after infection with Flavobactrium columnare (F. cloumnare) and grass carp reovirus II (GCRV II). The recombinant CiIL-20L produced in E. coli cells was shown to be effective in inducing the expression of Th cytokine genes (IFN-γ, IL-4/13A, IL-4/13B and IL-10), macrophage marker genes (arginase 2, IRF4, KLF4 and SOCS3) and inflammatory genes (IL-1ß, IL-6, IL-8 and TNFα) in the head kidney leukocytes when stimulated at 12 h. Long term culture (6 days) of head kidney macrophages in the presence of CiIL-20L leads to high expression of IRF4, TGFß1 and arginase 2. Our data suggest that IL-20 may play regulatory roles in promoting Th responses, macrophage differentiation and inflammation.

Feline Interleukin-31 Shares Overlapping Epitopes with the Oncostatin M Receptor and IL-31RA.

Interleukin-31 (IL-31) is a major protein involved in severe inflammatory skin disorders. Its signaling pathway is mediated through two type I cytokine receptors, IL-31RA (also known as the gp130-like receptor) and the oncostatin M receptor (OSMR). Understanding molecular details in these interactions would be helpful for developing antagonist anti-IL-31 monoclonal antibodies (mAbs) as potential therapies. Previous studies suggest that human IL-31 binds to IL-31RA and then recruits OSMR to form a ternary complex. In this model, OSMR cannot interact with IL-31 in the absence of IL-31RA. In this work, we show that feline IL-31 (fIL-31) binds independently with feline OSMR using surface plasmon resonance, an enzyme-linked immunosorbent assay, and yeast surface display. Moreover, competition experiments suggest that OSMR shares a partially overlapping epitope with IL-31RA. We then used deep mutational scanning to map the binding sites of both receptors on fIL-31. In agreement with previous studies of the human homologue, the binding site for IL31-RA contains fIL-31 positions E20 and K82, while the binding site for OSMR comprises the "PADNFERK" motif (P103-K110) and position G38. However, our results also revealed a new overlapping site, composed of positions R69, R72, P73, D76, D81, and E97, between both receptors that we called the "shared site". The conformational epitope of an anti-feline IL-31 mAb that inhibits both OSMR and IL-31RA also mapped to this shared site. Combined, our results show that fIL-31 binds IL-31RA and OSMR independently through a partially shared epitope. These results suggest reexamination of the putative canonical mechanisms for IL-31 signaling in higher animals.

Site-Specific Protein Labeling with Fluorophores as a Tool To Monitor Protein Turnover.

Proteins that terminally fail to acquire their native structure are detected and degraded by cellular quality control systems. Insights into cellular protein quality control are key to a better understanding of how cells establish and maintain the integrity of their proteome and of how failures in these processes cause human disease. Here we have used genetic code expansion and fast bio-orthogonal reactions to monitor protein turnover in mammalian cells through a fluorescence-based assay. We have used immune signaling molecules (interleukins) as model substrates and shown that our approach preserves normal cellular quality control, assembly processes, and protein functionality and works for different proteins and fluorophores. We have further extended our approach to a pulse-chase type of assay that can provide kinetic insights into cellular protein behavior. Taken together, this study establishes a minimally invasive method to investigate protein turnover in cells as a key determinant of cellular homeostasis.

Structure-based glycoengineering of interferon lambda 4 enhances its productivity and anti-viral potency.

Interferon lambda 4 (IFNλ4) has been recently known and studied for its role in hepatitis C virus (HCV) infection, but its clinical potential is significantly hampered due to its poor expression in vitro. Our study reports the successful production of IFNλ4 from a mammalian cell line through a glycoengineering and structure-based approach. We introduced de novo N-glycosylation of IFNλ4, guided by structural analysis, and produced IFNλ4 variants in Expi293F that displayed improved expression and potency. To preserve the structure and functionality of IFNλ4, the model structure of the IFNλ4 signaling complex was analyzed and the N-glycosylation candidate sites were selected. The receptor binding activity of engineered IFNλ4 variants and their receptor-mediated signaling pathway were similar to the E. coli version of IFNλ4 (eIFNλ4), while the antiviral activity and induction levels of interferon-stimulated gene (ISG) were all more robust in our variants. Our engineered IFNλ4 variants may be further developed for clinical applications and utilized in basic research to decipher the immunological roles of IFNλ4.

Potential of Flavonoid-Inspired Phytomedicines against COVID-19.

Flavonoids are widely used as phytomedicines. Here, we report on flavonoid phytomedicines with potential for development into prophylactics or therapeutics against coronavirus disease 2019 (COVID-19). These flavonoid-based phytomedicines include: caflanone, Equivir, hesperetin, myricetin, and Linebacker. Our in silico studies show that these flavonoid-based molecules can bind with high affinity to the spike protein, helicase, and protease sites on the ACE2 receptor used by the severe acute respiratory syndrome coronavirus 2 to infect cells and cause COVID-19. Meanwhile, in vitro studies show potential of caflanone to inhibit virus entry factors including, ABL-2, cathepsin L, cytokines (IL-1ß, IL-6, IL-8, Mip-1α, TNF-α), and PI4Kiiiß as well as AXL-2, which facilitates mother-to-fetus transmission of coronavirus. The potential for the use of smart drug delivery technologies like nanoparticle drones loaded with these phytomedicines to overcome bioavailability limitations and improve therapeutic efficacy are discussed.

Plasma Concentrations of Myeloid-Derived Growth Factor in Healthy Individuals and Patients with Acute Myocardial Infarction as Assessed by Multiple Reaction Monitoring-Mass Spectrometry.

Myeloid-derived growth factor (MYDGF in humans, Mydgf in mice) is a secreted protein with previously unknown biological functions. In a recent study, Mydgf was shown to mediate cardiac repair after acute myocardial infarction (MI) in mice. Lack of a sensitive assay to measure MYDGF in the circulation has hampered its further investigation. Here, we developed a liquid chromatography/multiple reaction monitoring-mass spectrometry MYDGF assay, employing SDS-PAGE-based protein fractionation to deplete high-abundant proteins and a stable isotope-labeled synthetic standard peptide for quantification. The assay had a lower limit of quantification of 0.8 ng/mL and a linear range up to 190 ng/mL. Within-run and total imprecision ranged from 8 to 17% and 11 to 20%, respectively. MYDGF plasma concentrations were not affected by either storage at room temperature for 4 h or up to three freeze-thaw cycles. Apparently healthy adults presented with a median (range) MYDGF concentration of 3.3 (1.3-6.7) ng/mL ( n = 120). MYDGF concentrations were elevated 2.7-fold ( P < 0.001) in patients with acute MI ( n = 101) and were associated with inflammatory biomarkers, renal dysfunction, and long-term cardiovascular mortality. The new assay and the favorable preanalytic characteristics of the analyte will facilitate studies into the pathophysiology of MYDGF and its potential use as a biomarker or protein therapeutic in patients with acute MI or other disease states.

Functional characterisation of interleukin 34 in grass carp Ctenopharyngodon idella.

Interleukin (IL) 34 plays an important role in regulating macrophage functions and inflammation process. IL-34 homologues have recently been discovered in fish but the functions have not been studied. In this study, an IL-34 homologue was identified in grass carp Ctenopharyngodon idella and its bioactivities were investigated. The grass carp IL-34 was constitutively expressed in tissues, with the highest expression detected in spleen. It could be up-regulated in spleen after infection with F. cloumnare and grass carp reovirus II, and in primary head kidney leucocytes by recombinant IL-4/13B. The recombinant IL-34 produced in bacteria and HEK293T cells showed stimulatory effect on the expression of IL-1ß, IL-6 and IL-8 but inhibited expression of IL-10 and TGF-ß1 in primary head kidney macrophages. The results demonstrate that IL-34 is a proinflammatory cytokine in grass carp.

The interleukin-like epithelial-mesenchymal transition inducer ILEI exhibits a non-interleukin-like fold and is active as a domain-swapped dimer.

Production and secretion of pro-metastatic proteins is a feature of many tumor cells. The FAM3C interleukin-like epithelial-to-mesenchymal-transition (EMT) inducer (ILEI) has been shown to be strongly up-regulated in several cancers and to be essential for tumor formation and metastasis in epithelial cells, correlating with a significant decrease in overall survival in colon and breast cancer patients. ILEI has been seen to interact with the γ-secretase presenilin 1 subunit (PS1). However, not much is known about the mechanism-of-action or the detailed ILEI structure. We present here the crystal structures of FAM3C ILEI and show that it exists as monomers but also as covalent dimers. The observed ILEI ß-ß-α fold confirmed previous indications that the FAM3C proteins do not form classical four-helix-bundle structures as was initially predicted. This provides the first experimental evidence that the interleukin-like EMT inducers are not evolutionarily related to the interleukins. However, more surprisingly, the ILEI dimer structure was found to feature a trans-linked domain swap, converting an intramolecular disulfide to intermolecular. Interestingly, dimeric but not monomeric ILEI was subsequently found to cause a dose-dependent increase in EpRas cell invasiveness comparable with TGF-ß, indicating that the dimer might be the active ILEI species. This is in line with a parallel study showing that covalent oligomerization of ILEI is essential for EMT and tumor progression in vivo The structures and the activity data give some first insight into the relationship between dimerization and ILEI function as well as indicate an intriguing link between ILEI, the PS1-protease, TGF-ß, and the TGF-ß receptor 1.

Emerging role of interleukin-31 and interleukin-31 receptor in pruritus in atopic dermatitis.

Atopic dermatitis (AD) is a chronic or chronically relapsing, eczematous, severely pruritic skin disorder associated with skin barrier dysfunction. The lesional skin of AD exhibits T helper 2 (TH 2)-deviated immune reactions. Interleukin-31 (IL-31), preferentially produced from TH 2 cells, is a potent pruritogenic cytokine, and its systemic and local administration induces scratching behavior in rodents, dogs and monkeys. Recent clinical trials have revealed that administration of an anti-IL-31 receptor antibody significantly alleviates pruritus in patients with AD. In this review, we summarize recent topics related to IL-31 and its receptor with special references to atopic itch.

A novel JNK is involved in immune response by regulating IL expression in oyster Crassostrea gigas.

The c-Jun N-terminal kinase (JNK) is a member of mitogen-activated protein kinases (MAPK) highly conserved from yeast to mammals and participates in regulating many physiological and pathological processes. In the present study, a novel JNK was identified from oyster Crassostrea gigas (designated as CgJNK) and its biological functions were investigated in response against lipopolysaccharide (LPS) stimulation. The CgJNK consists of 415 amino acids, which includes a serine/threonine protein kinase (S_TKc) domain with a conserved Thr-Pro-Tyr (TPY) motif. Phylogenetic analysis revealed that CgJNK shared high similarity with other members of the JNK subfamily. CgJNK mRNA was detected in all the tested tissues and CgJNK mRNA expression levels in hemocytes were significantly up-regulated from 6 to 72 h after LPS stimulation and reached the highest level (16.1-fold, p < 0.01) at 24 h. The phosphorylation level of CgJNK in C. gigas hemocytes was increased at 2 h after LPS stimulation. The subcellular localization of CgJNK phosphorylation in hemocytes was analyzed after LPS stimulation, and CgJNK phosphorylation could be detected in both cytoplasm and nucleus of oyster hemocytes at 2 h post LPS stimulation. Additionally, the interleukins (CgILs) were detected in hemocytes of CgJNK-knockdown oysters. CgIL17-1, CgIL17-2, CgIL17-4 and CgIL17-6 transcripts were decreased significantly in CgJNK-knockdown oysters at 24 h post LPS stimulation. In summary, these results suggested that CgJNK played an important role in the immune response of oysters by regulating IL expression.

Molecular characterization and expression analysis of large yellow croaker (Larimichthys crocea) interleukin-12A, 16 and 34 after poly I:C and Vibrio anguillarum challenge.

Interleukin-12, 16 and 34 are important pro-inflammatory cytokines, some of the most important components of the innate immunity system. Herein, we identified interleukin-12A (lcIL12A), 16 (lcIL16) and 34 (lcIL34) in large yellow croaker (Larimichthys crocea), and determined their expression profile in unchallenged and challenged tissues. The coding sequence (CDS) of lcIL12A comprised 600 bp long encoding a protein of 199 amino acids (aa), the CDS of lcIL16 was 2454 bp encoding a protein of 817 aa, and the CDS of lcIL34 was 657 bp encoding a protein of 267 aa. Phylogenetic analysis revealed similar results to homology comparison that lcIL12A was closest to IL12A of Dicentrarchus labrax (73%) and Serola dumerili (73%), while lcIL16 had the closest relation to Lates calcarofer (72.6%), and lcIL34 to Sparus aurata (88.9%). Multiple sequence alignment showed these interleukins were highly conserved with other vertebrate interleukins in their functional domains. Further, quantitative real time PCR (qPCR) analysis revealed that lcIL12A, lcIL16 and lcIL34 were constitutively expressed in all examined tissues, with significantly higher expression in spleen, liver and kidney. This was especially true for lcIL34 gene. Importantly, when challenged with polyinosinic:polycytidylic acid (poly I:C) and Vibrio anguillarum (V. anguillarum), the mRNA expressions of these interleukins were up-regulated in liver, spleen and kidney. Their top values got over 4 folds at least relative to their expression at time 0, and even lcIL12 reached 13.37 fold at 12-h point in spleen. These suggested their anti-viral and anti-bacterial roles and their involvement in the innate immune response of Larimichthys crocea. These results would have major implications in improving our understanding of the functions of interleukins to defend against pathogen infections in teleost species.