Insights into cytokine-receptor interactions from cytokine engineering.

Cytokines exert a vast array of immunoregulatory actions critical to human biology and disease. However, the desired immunotherapeutic effects of native cytokines are often mitigated by toxicity or lack of efficacy, either of which results from cytokine receptor pleiotropy and/or undesired activation of off-target cells. As our understanding of the structural principles of cytokine-receptor interactions has advanced, mechanism-based manipulation of cytokine signaling through protein engineering has become an increasingly feasible and powerful approach. Modified cytokines, both agonists and antagonists, have been engineered with narrowed target cell specificities, and they have also yielded important mechanistic insights into cytokine biology and signaling. Here we review the theory and practice of cytokine engineering and rationalize the mechanisms of several engineered cytokines in the context of structure. We discuss specific examples of how structure-based cytokine engineering has opened new opportunities for cytokines as drugs, with a focus on the immunotherapeutic cytokines interferon, interleukin-2, and interleukin-4.

Structural basis of cytokine-mediated activation of ALK family receptors.

Anaplastic lymphoma kinase (ALK)1 and the related leukocyte tyrosine kinase (LTK)2 are recently deorphanized receptor tyrosine kinases3. Together with their activating cytokines, ALKAL1 and ALKAL24-6 (also called FAM150A and FAM150B or AUGß and AUGα, respectively), they are involved in neural development7, cancer7-9 and autoimmune diseases10. Furthermore, mammalian ALK recently emerged as a key regulator of energy expenditure and weight gain11, consistent with a metabolic role for Drosophila ALK12. Despite such functional pleiotropy and growing therapeutic relevance13,14, structural insights into ALK and LTK and their complexes with cognate cytokines have remained scarce. Here we show that the cytokine-binding segments of human ALK and LTK comprise a novel architectural chimera of a permuted TNF-like module that braces a glycine-rich subdomain featuring a hexagonal lattice of long polyglycine type II helices. The cognate cytokines ALKAL1 and ALKAL2 are monomeric three-helix bundles, yet their binding to ALK and LTK elicits similar dimeric assemblies with two-fold symmetry, that tent a single cytokine molecule proximal to the cell membrane. We show that the membrane-proximal EGF-like domain dictates the apparent cytokine preference of ALK. Assisted by these diverse structure-function findings, we propose a structural and mechanistic blueprint for complexes of ALK family receptors, and thereby extend the repertoire of ligand-mediated dimerization mechanisms adopted by receptor tyrosine kinases.

Mechanism for the activation of the anaplastic lymphoma kinase receptor.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase (RTK) that regulates important functions in the central nervous system1,2. The ALK gene is a hotspot for chromosomal translocation events that result in several fusion proteins that cause a variety of human malignancies3. Somatic and germline gain-of-function mutations in ALK were identified in paediatric neuroblastoma4-7. ALK is composed of an extracellular region (ECR), a single transmembrane helix and an intracellular tyrosine kinase domain8,9. ALK is activated by the binding of ALKAL1 and ALKAL2 ligands10-14 to its ECR, but the lack of structural information for the ALK-ECR or for ALKAL ligands has limited our understanding of ALK activation. Here we used cryo-electron microscopy, nuclear magnetic resonance and X-ray crystallography to determine the atomic details of human ALK dimerization and activation by ALKAL1 and ALKAL2. Our data reveal a mechanism of RTK activation that allows dimerization by either dimeric (ALKAL2) or monomeric (ALKAL1) ligands. This mechanism is underpinned by an unusual architecture of the receptor-ligand complex. The ALK-ECR undergoes a pronounced ligand-induced rearrangement and adopts an orientation parallel to the membrane surface. This orientation is further stabilized by an interaction between the ligand and the membrane. Our findings highlight the diversity in RTK oligomerization and activation mechanisms.

Papain-like protease regulates SARS-CoV-2 viral spread and innate immunity.

The papain-like protease PLpro is an essential coronavirus enzyme that is required for processing viral polyproteins to generate a functional replicase complex and enable viral spread1,2. PLpro is also implicated in cleaving proteinaceous post-translational modifications on host proteins as an evasion mechanism against host antiviral immune responses3-5. Here we perform biochemical, structural and functional characterization of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) PLpro (SCoV2-PLpro) and outline differences with SARS-CoV PLpro (SCoV-PLpro) in regulation of host interferon and NF-κB pathways. SCoV2-PLpro and SCoV-PLpro share 83% sequence identity but exhibit different host substrate preferences; SCoV2-PLpro preferentially cleaves the ubiquitin-like interferon-stimulated gene 15 protein (ISG15), whereas SCoV-PLpro predominantly targets ubiquitin chains. The crystal structure of SCoV2-PLpro in complex with ISG15 reveals distinctive interactions with the amino-terminal ubiquitin-like domain of ISG15, highlighting the high affinity and specificity of these interactions. Furthermore, upon infection, SCoV2-PLpro contributes to the cleavage of ISG15 from interferon responsive factor 3 (IRF3) and attenuates type I interferon responses. Notably, inhibition of SCoV2-PLpro with GRL-0617 impairs the virus-induced cytopathogenic effect, maintains the antiviral interferon pathway and reduces viral replication in infected cells. These results highlight a potential dual therapeutic strategy in which targeting of SCoV2-PLpro can suppress SARS-CoV-2 infection and promote antiviral immunity.

Treatment of type 2 diabetes with the designer cytokine IC7Fc.

The gp130 receptor cytokines IL-6 and CNTF improve metabolic homeostasis but have limited therapeutic use for the treatment of type 2 diabetes. Accordingly, we engineered the gp130 ligand IC7Fc, in which one gp130-binding site is removed from IL-6 and replaced with the LIF-receptor-binding site from CNTF, fused with the Fc domain of immunoglobulin G, creating a cytokine with CNTF-like, but IL-6-receptor-dependent, signalling. Here we show that IC7Fc improves glucose tolerance and hyperglycaemia and prevents weight gain and liver steatosis in mice. In addition, IC7Fc either increases, or prevents the loss of, skeletal muscle mass by activation of the transcriptional regulator YAP1. In human-cell-based assays, and in non-human primates, IC7Fc treatment results in no signs of inflammation or immunogenicity. Thus, IC7Fc is a realistic next-generation biological agent for the treatment of type 2 diabetes and muscle atrophy, disorders that are currently pandemic.

Recognition of Lys48-Linked Di-ubiquitin and Deubiquitinating Activities of the SARS Coronavirus Papain-like Protease.

Deubiquitinating enzymes (DUBs) recognize and cleave linkage-specific polyubiquitin (polyUb) chains, but mechanisms underlying specificity remain elusive in many cases. The severe acute respiratory syndrome (SARS) coronavirus papain-like protease (PLpro) is a DUB that cleaves ISG15, a two-domain Ub-like protein, and Lys48-linked polyUb chains, releasing diUb(Lys48) products. To elucidate this specificity, we report the 2.85 Å crystal structure of SARS PLpro bound to a diUb(Lys48) activity-based probe. SARS PLpro binds diUb(Lys48) in an extended conformation via two contact sites, S1 and S2, which are proximal and distal to the active site, respectively. We show that specificity for polyUb(Lys48) chains is predicated on contacts in the S2 site and enhanced by an S1-S1' preference for a Lys48 linkage across the active site. In contrast, ISG15 specificity is dominated by contacts in the S1 site. Determinants revealed for polyUb(Lys48) specificity should prove useful in understanding PLpro deubiquitinating activities in coronavirus infections.

A new look at cytokine signaling.

Signal transduction is initiated when a cytokine binds to the extracellular domains of its receptors, bringing them together and triggering a complicated sequence of events inside the cell. In this issue, LaPorte et al. (2008) present crystal structures of three signaling complexes of the cytokines interleukin-4 and interleukin-13 with their receptors, showing how events taking place outside the cell may affect the specificity of signal transduction.

Engineering a potent receptor superagonist or antagonist from a novel IL-6 family cytokine ligand.

Interleukin-6 (IL-6) family cytokines signal through multimeric receptor complexes, providing unique opportunities to create novel ligand-based therapeutics. The cardiotrophin-like cytokine factor 1 (CLCF1) ligand has been shown to play a role in cancer, osteoporosis, and atherosclerosis. Once bound to ciliary neurotrophic factor receptor (CNTFR), CLCF1 mediates interactions to coreceptors glycoprotein 130 (gp130) and leukemia inhibitory factor receptor (LIFR). By increasing CNTFR-mediated binding to these coreceptors we generated a receptor superagonist which surpassed the potency of natural CNTFR ligands in neuronal signaling. Through additional mutations, we generated a receptor antagonist with increased binding to CNTFR but lack of binding to the coreceptors that inhibited tumor progression in murine xenograft models of nonsmall cell lung cancer. These studies further validate the CLCF1-CNTFR signaling axis as a therapeutic target and highlight an approach of engineering cytokine activity through a small number of mutations.

Functional characterisation of two receptor interaction determinants in human thymic stromal lymphopoietin.

Thymic stromal lymphopoietin (TSLP) is a pro-inflammatory cytokine with important pathological roles in Asthma bronchiale, malignant tumours and other diseases. The heterodimeric human TSLP receptor (hTSLPR) consists of the TSLP-binding subunit (TSLPRα) and the IL-7Rα-subunit. We studied the properties of hTSLP variants with mutations in their bipartite interaction interface towards IL-7Rα. One mutant (T46D/K101D) showed only mild impairment in receptor affinity but a massive reduction in biological activity. To facilitate the future development of hTSLP mutants with drug properties, we have devised a eukaryontic cytokine display assay with activity read-out and intrinsic genotype-phenotype coupling.

Antibodies against the erythroferrone N-terminal domain prevent hepcidin suppression and ameliorate murine thalassemia.

Erythroferrone (ERFE) is produced by erythroblasts in response to erythropoietin (EPO) and acts in the liver to prevent hepcidin stimulation by BMP6. Hepcidin suppression allows for the mobilization of iron to the bone marrow for the production of red blood cells. Aberrantly high circulating ERFE in conditions of stress erythropoiesis, such as in patients with ß-thalassemia, promotes the tissue iron accumulation that substantially contributes to morbidity in these patients. Here we developed antibodies against ERFE to prevent hepcidin suppression and to correct the iron loading phenotype in a mouse model of ß-thalassemia [Hbb(th3/+) mice] and used these antibodies as tools to further characterize ERFE's mechanism of action. We show that ERFE binds to BMP6 with nanomolar affinity and binds BMP2 and BMP4 with somewhat weaker affinities. We found that BMP6 binds the N-terminal domain of ERFE, and a polypeptide derived from the N terminus of ERFE was sufficient to cause hepcidin suppression in Huh7 hepatoma cells and in wild-type mice. Anti-ERFE antibodies targeting the N-terminal domain prevented hepcidin suppression in ERFE-treated Huh7 cells and in EPO-treated mice. Finally, we observed a decrease in splenomegaly and serum and liver iron in anti-ERFE-treated Hbb(th3/+) mice, accompanied by an increase in red blood cells and hemoglobin and a decrease in reticulocyte counts. In summary, we show that ERFE binds BMP6 directly and with high affinity, and that antibodies targeting the N-terminal domain of ERFE that prevent ERFE-BMP6 interactions constitute a potential therapeutic tool for iron loading anemias.

Assessment of tear inflammatory cytokines concentration in patients with diabetes with varying severity of involvement.

Diabetic retinopathy (DR), as one of the most important causes of blindness in Western societies, is a common micro-vasculopathy associated with diabetes. There is growing evidence of the role of inflammation in its development. This study was designed to measure cytokines in patients with diabetes with different stages of retinopathy .In this study, tear concentrations of three types of cytokines with different angiogenic properties including IL-1RA, IL-8, and TNF-α were measured in patients with diabetes without retinopathy, with non-proliferative retinopathy, with proliferative retinopathy, and in a healthy control group. The results showed that concentrations of TNF-α and IL-8 were higher in the tear sample of diabetics than in the control group and the concentrations of these cytokines were higher in patients with more advanced stages of diabetes, while the tear level of IL-1RA was significantly lower in diabetics. Based on these findings, it can be concluded that diabetes and its progression of severity affects the tear levels of IL-1RA, IL-8, and TNF-α inflammatory cytokines.

Destabilization of the human RED-SMU1 splicing complex as a basis for host-directed antiinfluenza strategy.

New therapeutic strategies targeting influenza are actively sought due to limitations in current drugs available. Host-directed therapy is an emerging concept to target host functions involved in pathogen life cycles and/or pathogenesis, rather than pathogen components themselves. From this perspective, we focused on an essential host partner of influenza viruses, the RED-SMU1 splicing complex. Here, we identified two synthetic molecules targeting an α-helix/groove interface essential for RED-SMU1 complex assembly. We solved the structure of the SMU1 N-terminal domain in complex with RED or bound to one of the molecules identified to disrupt this complex. We show that these compounds inhibiting RED-SMU1 interaction also decrease endogenous RED-SMU1 levels and inhibit viral mRNA splicing and viral multiplication, while preserving cell viability. Overall, our data demonstrate the potential of RED-SMU1 destabilizing molecules as an antiviral therapy that could be active against a wide range of influenza viruses and be less prone to drug resistance.

Micro- and nanosensors for detecting blood pathogens and biomarkers at different points of sepsis care.

Severe infections can cause a dysregulated response leading to organ dysfunction known as sepsis. Sepsis can be lethal if not identified and treated right away. This requires measuring biomarkers and pathogens rapidly at the different points where sepsis care is provided. Current commercial approaches for sepsis diagnosis are not fast, sensitive, and/or specific enough for meeting this medical challenge. In this article, we review recent advances in the development of diagnostic tools for sepsis management based on micro- and nanostructured materials. We start with a brief introduction to the most popular biomarkers for sepsis diagnosis (lactate, procalcitonin, cytokines, C-reactive protein, and other emerging protein and non-protein biomarkers including miRNAs and cell-based assays) and methods for detecting bacteremia. We then highlight the role of nano- and microstructured materials in developing biosensors for detecting them taking into consideration the particular needs of every point of sepsis care (e.g., ultrafast detection of multiple protein biomarkers for diagnosing in triage, emergency room, ward, and intensive care unit; quantitative detection to de-escalate treatment; ultrasensitive and culture-independent detection of blood pathogens for personalized antimicrobial therapies; robust, portable, and web-connected biomarker tests outside the hospital). We conclude with an overview of the most utilized nano- and microstructured materials used thus far for solving issues related to sepsis diagnosis and point to new challenges for future development.

Structural Characterization of the Interaction between the αMI-Domain of the Integrin Mac-1 (αMß2) and the Cytokine Pleiotrophin.

Integrin Mac-1 (αMß2) is an adhesion receptor vital to many functions of myeloid leukocytes. It is also the most promiscuous member of the integrin family capable of recognizing a broad range of ligands. In particular, its ligand-binding αMI-domain is known to bind cationic proteins/peptides depleted in acidic residues. This contradicts the canonical ligand-binding mechanism of αI-domains, which requires an acidic amino acid in the ligand to coordinate the divalent cation within the metal ion-dependent adhesion site (MIDAS) of αI-domains. The lack of acidic amino acids in the αMI-domain-binding sequences suggests the existence of an as-yet uncharacterized interaction mechanism. In the present study, we analyzed interactions of the αMI-domain with a representative Mac-1 ligand, the cationic cytokine pleiotrophin (PTN). Through NMR chemical shift perturbation analysis, cross saturation, NOESY, and mutagenesis studies, we found the interaction between the αMI-domain and PTN is divalent cation-independent and mediated mostly by hydrophobic contacts between the N-terminal domain of PTN and residues in the α5-ß5 loop of αMI-domain. The observation that increased ionic strength weakens the interaction between the proteins indicates electrostatic forces may also play a significant role in the binding. On the basis of the results from these experiments, we formulated a model of the interaction between the αMI-domain and PTN.

Enzymatic Synthesis of 3'-5', 3'-5' Cyclic Dinucleotides, Their Binding Properties to the Stimulator of Interferon Genes Adaptor Protein, and Structure/Activity Correlations.

The 3'-5', 3'-5' cyclic dinucleotides (3'3'CDNs) are bacterial second messengers that can also bind to the stimulator of interferon genes (STING) adaptor protein in vertebrates and activate the host innate immunity. Here, we profiled the substrate specificity of four bacterial dinucleotide synthases from Vibrio cholerae (DncV), Bacillus thuringiensis (btDisA), Escherichia coli (dgcZ), and Thermotoga maritima (tDGC) using a library of 33 nucleoside-5'-triphosphate analogues and then employed these enzymes to synthesize 24 3'3'CDNs. The STING affinity of CDNs was evaluated in cell-based and biochemical assays, and their ability to induce cytokines was determined by employing human peripheral blood mononuclear cells. Interestingly, the prepared heterodimeric 3'3'CDNs bound to the STING much better than their homodimeric counterparts and showed similar or better potency than bacterial 3'3'CDNs. We also rationalized the experimental findings by in-depth STING-CDN structure-activity correlations by dissecting computed interaction free energies into a set of well-defined and intuitive terms. To this aim, we employed state-of-the-art methods of computational chemistry, such as quantum mechanics/molecular mechanics (QM/MM) calculations, and complemented the computed results with the {STING:3'3'c-di-ara-AMP} X-ray crystallographic structure. QM/MM identified three outliers (mostly homodimers) for which we have no clear explanation of their impaired binding with respect to their heterodimeric counterparts, whereas the R2 = 0.7 correlation between the computed ΔG'int_rel and experimental ΔTm's for the remaining ligands has been very encouraging.

Inference of molecular structure for characterization and improvement of clinical grade immunocytokines.

The use of immunomodulatory agents for the treatment of cancer is gaining a growing biopharmaceutical interest. Antibody-cytokine fusion proteins, namely immunocytokines, represent a promising solution for the regulation of the immune system at the site of disease. The three-dimensional arrangement of these molecules can profoundly influence their biological activity and pharmacokinetic properties. Structural techniques might provide important insight in the 3D arrangement of immunocytokines. Here, we performed structure investigations on clinical grade fusion proteins L19-IL2, IL12-L19L19 and L19L19-IL2 to elucidate their quaternary organization. Crystallographic characterization of the common L19 antibody fragment at a resolution of 2.0-Å was combined with low-resolution studies of the full-length chimeric molecules using small-angle synchrotron X-ray scattering (SAXS) and negative stain electron microscopy. Characterization of the full-length quaternary structures of the immunocytokines in solution by SAXS consistently supported the diabody structure in the L19-IL2 immunocytokine and allowed generation of low-resolution models of the chimeric proteins L19L19-IL2 and IL12-L19L19. Comparison with 3D reconstructions obtained from negative-stain electron microscopy revealed marked flexibility associated to the linker regions connecting the cytokine and the antibody components of the chimeric proteins. Collectively, our results indicate that low-resolution molecular structure characterizations provide useful complementary insights for the quality control of immunocytokines, constituting a powerful tool to guide the design and the subsequent optimization steps towards clinical enhancement of these chimeric protein reagents.

The role of pro-domains in human growth factors and cytokines.

Many growth factors and cytokines are produced as larger precursors, containing pro-domains, that require proteolytic processing to release the bioactive ligand. These pro-domains can be significantly larger than the mature domains and can play an active role in the regulation of the ligands. Mining the UniProt database, we identified almost one hundred human growth factors and cytokines with pro-domains. These are spread across several unrelated protein families and vary in both their size and composition. The precise role of each pro-domain varies significantly between the protein families. Typically they are critical for controlling bioactivity and protein localisation, and they facilitate diverse mechanisms of activation. Significant gaps in our understanding remain for pro-domain function - particularly their fate once the bioactive ligand has been released. Here we provide an overview of pro-domain roles in human growth factors and cytokines, their processing, regulation and activation, localisation as well as therapeutic potential.

Characterization of a common carp intelectin gene with bacterial binding and agglutination activity.

Intelectin (ITLN) is a type of glycan-binding lectin involved in many physiological processes and some human diseases. Here we report a common carp intelectin (cITLN). Like other orthologs, cITLN also contains a conserved fibrinogen-related domain (FReD) and a unique intelectin domain, expresses in all the tissues tested with the highest level in the hindgut, and responds to bacterial challenge in the acute phase. We also expressed cITLN in Escherichia coli (E. coli) system, and the purified recombinant cITLN could neither affect the surface of bacteria nor inhibit the growth of bacteria, but it can agglutinate both gram-positive and gram-negative bacteria in a calcium-dependent manner. The cITLN's ability of agglutination of gram-positive bacteria is stronger than that of gram-negative bacteria. This is probably because recombinant cITLN could binding peptidoglycan (PGN) with a higher degree to lipopolysaccharide (LPS). Our results of cITLN provided new insight into the function of intelectin in the intestinal mucosal immunity.

Effects of empagliflozin on proinflammatory cytokines and other coronary risk factors in patients with type 2 diabetes mellitus: A single-arm real-world observation.

OBJECTIVE: This single-arm real-world observation aims to examine the effects of empagliflozin (EMPA) on coronary risk factors among subjects with known diabetes. MATERIALS AND METHODS: Records of 63 subjects with type 2 diabetes mellitus, receiving EMPA were drawn for this study. Of 63 patients with diabetes, 6 were excluded, and the remaining 57 received EMPA (25 mg/day) for 24 weeks. Clinical data, dietary intakes, and physical activity were assessed by validated questionnaires. RESULTS: Treatment with EMPA was associated with significant decline in fasting and 2-hour post-prandial blood glucose and Hb1c indicating that this agent has potential antidiabetic effects. Pro-inflammatory cytokines; C-reactive protein, TNF-α, and interleukin-6 showed significant reduction after treatment with EMPA, compared to baseline levels. Apart from these changes, parameters of oxidative stress, thiobarbituric acid reactive substances, malondialdehyde, and diene conjugates as well as uric acid, showed a significant decline with an increase in antioxidant vitamins A, E, and C and beta-carotene as well as nitrite. There was a significant decline in serum uric acid, systolic and diastolic blood pressures, and angiotensin-converting enzyme (ACE), with a non-significant reduction in body weight and body mass index as well as in waist circumference of modest significance, after intervention of 12 weeks compared to baseline levels. Total cholesterol, VLDL cholesterol and triglycerides showed non-significant decline compared to baseline levels. CONCLUSION: It is possible that EMPA administration can cause a significant decline in pro-inflammatory cytokines along with blood glucose, Hb1c, oxidative stress, uric acid, blood pressures, and ACE with an increase in antioxidant vitamins and nitrite. Randomized, controlled intervention trials would be necessary to confirm our findings.

Reduced biomechanical models for precision-cut lung-slice stretching experiments.

Precision-cut lung-slices (PCLS), in which viable airways embedded within lung parenchyma are stretched or induced to contract, are a widely used ex vivo assay to investigate bronchoconstriction and, more recently, mechanical activation of pro-remodelling cytokines in asthmatic airways. We develop a nonlinear fibre-reinforced biomechanical model accounting for smooth muscle contraction and extracellular matrix strain-stiffening. Through numerical simulation, we describe the stresses and contractile responses of an airway within a PCLS of finite thickness, exposing the importance of smooth muscle contraction on the local stress state within the airway. We then consider two simplifying limits of the model (a membrane representation and an asymptotic reduction in the thin-PCLS-limit), that permit analytical progress. Comparison against numerical solution of the full problem shows that the asymptotic reduction successfully captures the key elements of the full model behaviour. The more tractable reduced model that we develop is suitable to be employed in investigations to elucidate the time-dependent feedback mechanisms linking airway mechanics and cytokine activation in asthma.