IL17F Human, sf9

What is IL17F and how is it produced in sf9 cells?

IL17F is a cytokine belonging to the IL-17 family that shares sequence similarity with IL17A. It is a biologically active protein expressed by activated T cells, mast cells, and basophils that plays crucial roles in inflammatory responses . In nature, IL17F exists as a homodimer and shows homology to herpes virus early protein . It is one of six members (IL-17A-F) of this cytokine family and is highly expressed by activated effector memory T cells .

When produced in sf9 insect cells, IL17F is expressed as a glycosylated polypeptide chain containing 142 amino acids (residues 31-163) with a molecular mass of approximately 16kDa, though it typically appears as 18-28 kDa on SDS-PAGE due to glycosylation . The recombinant protein is commonly expressed with a 9-amino acid His tag at the C-terminus, facilitating purification through proprietary chromatographic techniques . This baculovirus expression system allows for eukaryotic post-translational modifications, particularly glycosylation, which may be important for certain functional studies.

What are the structural characteristics of IL17F and how do they influence function?

IL17F has a distinct amino acid sequence: "ADPRKIPKVG HTFFQKPESC PPVPGGSMKL DIGIINENQR VSMSRNIESR STSPWNYTVT WDPNRYPSEV VQAQCRNLGC INAQGKEDIS MNSVPIQQET LVVRRKHQGC SVSFQLEKVL VTVGCTCVTP VIHHVQHHHH HH" (the final histidine residues represent the His-tag) . The protein adopts a homodimeric structure stabilized by disulfide bonds, which is critical for its biological activity.

The structural features of IL17F enable it to bind to the IL-17 receptor (IL-17R), initiating a signaling cascade that requires receptor ubiquitination by TRAF6 . This interaction activates the Raf1-mitogen-activated protein kinase 1/2-extracellular-regulated kinase 1/2-p90 ribosomal S6 kinase-cyclic AMP response element-binding protein signaling pathway, leading to the expression of IFN-gamma-inducible protein 10 (IP-10) . The specific structural elements of IL17F also allow it to form heterodimers with IL17A, which may have distinct biological activities compared to either homodimer .

How do IL17F expression conditions in CD4+ T cells compare with recombinant production?

In human CD4+ T cells, IL17F expression is naturally regulated by several factors. Studies have shown that IL-1β, IL-23, anti-CD3, and anti-CD28 mAb stimulation can induce both IL-17A and IL-17F expression in CD4+ T cells . Strong co-stimulation with anti-CD28 mAb increases IL-17F+IL-17A- and IL-17A+IL-17F+ CD4+ T cell frequencies, while IL-17A+IL-17F- CD4+ T cell frequencies decrease, partly through an IL-2-dependent mechanism .

In contrast, recombinant IL17F production in sf9 insect cells involves baculovirus-mediated expression of the protein with controlled conditions that maximize yield and purity . While natural IL17F expression in T cells is tightly regulated and dependent on the inflammatory microenvironment, recombinant production aims for constitutive high-level expression. This fundamental difference should be considered when using recombinant IL17F in experimental settings, as the protein may lack certain context-dependent modifications present in naturally produced cytokine.

What is the stability profile of IL17F Human sf9?

IL17F Human produced in sf9 cells is typically supplied as a sterile filtered colorless solution at a concentration of 0.5mg/ml in a buffer containing Phosphate Buffered Saline (pH 7.4), 20% glycerol, and 1mM DTT . The glycerol and DTT components enhance protein stability by preventing aggregation and maintaining the reduced state of cysteine residues, respectively.

For optimal stability, the protein should be stored according to the manufacturer's recommendations, typically at -80°C for long-term storage with minimal freeze-thaw cycles. The presence of glycerol in the formulation helps prevent damage during freeze-thaw cycles by reducing ice crystal formation. The purity of commercially available IL17F is generally greater than 95.0% as determined by SDS-PAGE , which contributes to its stability profile by minimizing the presence of proteases or other contaminants that could degrade the protein.

How do IL17F homodimers compare functionally to IL17A/F heterodimers?

IL17F can exist as homodimers or form heterodimers with IL17A . These different dimeric forms exhibit distinct biological properties and potencies in inflammatory responses. IL17F homodimers generally demonstrate lower pro-inflammatory activity compared to IL17A homodimers, with the IL17A/F heterodimer showing intermediate potency.

Flow cytometry analysis has identified three distinct IL-17-expressing CD4+ T cell populations: IL-17A+IL-17F- single-positive, IL-17F+IL-17A- single-positive, and IL-17A+IL-17F+ double-positive cells . These populations have different cytokine profiles: IL-17F+IL-17A- and IL-17A+IL-17F+ CD4+ T cells contain lower proportions of IL-10-expressing and GM-CSF-expressing cells but higher proportions of IFN-γ-expressing cells compared to IL-17A+IL-17F- CD4+ T cells . This suggests that cells producing IL17F, either alone or with IL17A, may promote more robust pro-inflammatory responses with less immunoregulatory capacity.

Research indicates that combined blockade of IL-17A and IL-17F is more effective at reducing inflammation than blockade of IL-17A alone , highlighting the distinct but complementary roles of these cytokines in driving inflammatory processes.

What are the differences in signaling pathways between IL17F and other IL17 family members?

IL17F and IL17A share signaling pathways but exhibit distinct functions in immune responses and diseases . Both cytokines signal through the IL-17 receptor A (IL-17RA), requiring tumor necrosis factor receptor–associated factor 6 (TRAF6) and Act1 for proinflammatory gene expression .

Despite these similarities, knockout studies have revealed important functional differences. IL-17, but not IL-17F, was required for the initiation of experimental autoimmune encephalomyelitis . In contrast, IL-17F-deficient mice, but not IL-17-deficient mice, showed defective airway neutrophilia in response to allergen challenge .

The differential roles extend to disease models: in asthma models, IL-17 deficiency reduced T helper type 2 responses, while IL-17F-deficient mice displayed enhanced type 2 cytokine production and eosinophil function . Additionally, IL-17F deficiency resulted in reduced colitis caused by dextran sulfate sodium, whereas IL-17 knockout mice developed more severe disease . These findings demonstrate that despite signaling through similar pathways, IL-17F functions differently than IL-17A in immune responses and diseases.

What experimental considerations should be made when designing IL17F functional assays?

When designing functional assays for IL17F, researchers should consider several important factors:

• Receptor expression: Ensure target cells express appropriate levels of IL-17R receptor complexes. IL17F signals through IL-17RA, but with different binding affinities compared to IL17A.

• Synergistic effects: IL17F exhibits strong synergy with TNF-α in promoting inflammatory gene expression . Consider including TNF-α in experimental designs to observe maximal responses.

• Readout selection: Appropriate readouts include measuring induced cytokines (IL-6, IL-8, GM-CSF), chemokines, or adhesion molecules in bronchial epithelial cells, vein endothelial cells, fibroblasts, and eosinophils .

• Recombinant protein source: The source of recombinant IL17F may affect activity. E.coli-derived recombinant human IL-17F is non-glycosylated , while sf9-derived IL17F is glycosylated , potentially affecting receptor interactions.

• Homodimer vs. heterodimer: Determine whether to study IL17F homodimers or IL17A/F heterodimers, as they may have distinct activities. Heterodimer formation can be achieved by co-expressing both proteins.

• Blocking strategies: When attempting to block IL17F function, consider that targeting IL17F alone may not completely abolish inflammatory responses due to redundancy with IL17A. Combined blockade of IL-17A and IL-17F is more effective at reducing inflammation than blockade of IL-17A alone .

How do IL17F knockout models reveal distinct functions from IL17A in disease states?

Knockout studies have provided critical insights into the distinct functions of IL17F compared to IL17A in various disease models:

• Autoimmune encephalomyelitis: IL-17A, but not IL-17F, was found to be required for the initiation of experimental autoimmune encephalomyelitis, suggesting a more critical role for IL17A in this neuroinflammatory condition .

• Airway inflammation: IL-17F-deficient mice, but not IL-17A-deficient mice, exhibited defective airway neutrophilia in response to allergen challenge, highlighting IL17F's specific role in neutrophil recruitment to the airways .

• Asthma models: While IL-17A deficiency reduced T helper type 2 responses, IL-17F-deficient mice displayed enhanced type 2 cytokine production and eosinophil function, suggesting IL17F may actually restrain certain aspects of type 2 inflammation in asthma .

• Colitis models: IL-17F deficiency resulted in reduced colitis severity caused by dextran sulfate sodium, whereas IL-17A knockout mice developed more severe disease . This indicates opposing roles for these cytokines in intestinal inflammation.

These findings demonstrate that despite structural similarities and shared receptor components, IL17F and IL17A play distinct and sometimes opposing roles in various inflammatory conditions. This functional diversity highlights the importance of specifically targeting individual IL17 family members in therapeutic approaches.

What are the optimal methods for detecting IL17F expression and secretion?

Researchers have several complementary techniques to detect IL17F expression and secretion:

• Flow cytometry: For intracellular detection of IL17F in individual cells, stimulate cells with PMA/ionomycin in the presence of Golgi-Stop for 3 hours, then perform intracellular staining . This technique allows simultaneous detection of IL17F with other cytokines and cell surface markers to characterize IL17F-producing cell populations.

• ELISA: For quantifying secreted IL17F in culture supernatants. Commercial IL17F-specific ELISAs can detect concentrations in the pg/mL range . When interpreting results, consider that different ELISA antibody affinities make it difficult to compare absolute levels of different cytokines.

• qRT-PCR: For measuring IL17F gene expression at the mRNA level. This is particularly useful for monitoring transcriptional regulation but does not necessarily correlate with protein secretion due to post-transcriptional regulation.

• Western blotting: For detecting IL17F protein in cell lysates or concentrated supernatants, using specific antibodies. This technique can distinguish between monomeric and dimeric forms under non-reducing and reducing conditions.

For optimal detection in experimental systems, stimulation of CD4+ T cells with IL-1β, IL-23, anti-CD3, and anti-CD28 mAb has been shown to effectively induce both IL-17A and IL-17F expression . In T cell/monocyte co-cultures, LPS stimulation can increase IL-17F+ CD4+ T cell frequencies by 3.4-fold and IL-17F secretion by 1.8-fold .

How can researchers differentiate between effects of IL17F versus other IL17 family members?

Distinguishing the specific contributions of IL17F from other IL17 family members in experimental systems requires several strategic approaches:

• Selective neutralization: Use highly specific neutralizing antibodies that target IL17F without cross-reactivity to other family members, particularly IL17A. Confirm specificity using recombinant proteins.

• Knockout/knockdown models: Utilize cells or animal models with selective knockout or knockdown of IL17F. Compare these to wild-type and IL17A knockout models to identify differential effects. Studies have shown distinct phenotypes between IL17F-deficient and IL17A-deficient mice in various disease models .

• Recombinant proteins: Apply purified recombinant IL17F versus IL17A or other family members at equimolar concentrations to determine differential responses. Consider using both homodimers and heterodimers to understand their distinct activities.

• Receptor blockade: Selectively block specific receptor components that may differentially affect signaling by different IL17 family members.

• Downstream signaling analysis: Monitor activation of signaling pathways that may be differentially regulated by IL17F versus other family members, such as the Raf1-MAPK1/2-ERK1/2-p90 RSK-CREB pathway that is specifically associated with IL17F-induced IP-10 expression .

• Transcriptional profiling: Perform RNA-seq or microarray analysis to identify gene signatures specifically regulated by IL17F versus other family members.

By combining these approaches, researchers can more confidently attribute observed effects to IL17F rather than other IL17 family members.

How is IL17F implicated in human inflammatory and autoimmune diseases?

IL17F has been implicated in several human inflammatory and autoimmune conditions:

• Inflammatory bowel disease: Intestinal IL17F gene expression is increased in active Crohn's disease, and IL-17A & IL-17F alleles influence the susceptibility to and pathophysiological features of ulcerative colitis independently .

• Functional dyspepsia: IL-17F and MIF gene polymorphisms are significantly associated with the development of functional dyspepsia .

• Allergic airway inflammation: IL-17F is involved in allergic airway inflammation and can induce several cytokines, chemokines, and adhesion molecules in bronchial epithelial cells . It was found in the airways of allergic asthma patients upon allergen challenge, and a mutation in the IL-17F gene was shown to be associated with human asthma and chronic obstructive pulmonary disease .

• Rheumatoid arthritis, psoriasis, and multiple sclerosis: Reports strongly suggest the involvement of IL-17 family cytokines, including IL17F, in these chronic inflammatory diseases .

Understanding IL17F's specific contributions to these conditions is important for developing targeted therapies. While IL17A-targeting therapies have shown success in conditions like psoriasis, targeting both IL17A and IL17F might provide additional benefits in certain inflammatory disorders where IL17F plays a distinct pathogenic role.

What are the key considerations when translating IL17F research from in vitro to in vivo models?

When translating IL17F research from in vitro studies to in vivo models, researchers should consider several important factors:

• Expression system differences: Recombinant IL17F produced in sf9 cells is glycosylated , while E.coli-derived IL17F is non-glycosylated . These differences may affect in vivo half-life, receptor binding, and biological activity. Consider which form best represents the native cytokine for your research question.

• Delivery methods: For in vivo overexpression studies, different delivery methods have been utilized, including adenoviral infection or lipofectamine-mediated gene transfer to acutely overexpress IL17F . These approaches may yield different expression patterns and biological effects.

• Model selection: Different animal models show varying responses to IL17F manipulation. For example, in asthma models, IL-17F deficiency resulted in enhanced type 2 cytokine production and eosinophil function, while in colitis models, IL-17F deficiency reduced disease severity . Select models most relevant to your specific research question.

• Physiological concentrations: Ensure that in vivo studies use physiologically relevant concentrations of IL17F. In human studies, IL17F secretion in stimulated T cell/monocyte co-cultures averaged 428 pg/mL after LPS stimulation .

• Cellular sources: In vitro studies may focus on a single cell type, while in vivo, IL17F can be produced by multiple cell types including activated T cells, mast cells, and basophils . Consider the complexity of these cellular interactions when designing in vivo experiments.

• Compensatory mechanisms: In vivo knockout studies have revealed potential compensatory mechanisms that are not apparent in vitro. For example, IL17F deficiency may lead to altered regulation of other inflammatory mediators that affect the disease phenotype.

By carefully considering these factors, researchers can design more translationally relevant studies that bridge the gap between in vitro findings and in vivo applications.

What emerging technologies can advance understanding of IL17F biology?

Several cutting-edge technologies can significantly advance our understanding of IL17F biology:

• Single-cell transcriptomics/proteomics: These technologies can reveal heterogeneity within IL17F-producing cell populations and identify novel subsets with distinct functional properties. They can also elucidate how individual cells respond to IL17F stimulation across different tissues.

• CRISPR-Cas9 genome editing: Precise modification of IL17F, receptor components, or downstream signaling molecules can provide insights into structure-function relationships and regulatory mechanisms. This approach can also be used to create improved cellular and animal models.

• Protein engineering: Creating modified versions of IL17F with altered receptor binding properties, stability, or dimerization capacity can help dissect functional domains and potentially develop IL17F variants with therapeutic properties.

• Advanced imaging techniques: Techniques like super-resolution microscopy and intravital imaging can visualize IL17F-receptor interactions and trafficking in real-time within living cells and tissues.

• Systems biology approaches: Integration of multi-omics data (genomics, transcriptomics, proteomics, metabolomics) can provide a comprehensive understanding of IL17F-mediated networks in different physiological and pathological contexts.

• Organoid and microphysiological systems: These advanced 3D culture systems can better recapitulate the complexity of tissue environments where IL17F functions, allowing for more physiologically relevant studies of its effects.

These technologies will help answer fundamental questions about IL17F biology, including its tissue-specific functions, regulatory mechanisms, and potential as a therapeutic target in various inflammatory conditions.