IL17F Rat

What is the molecular structure of rat IL-17F and how does it compare to other species?

Rat IL-17F is a 19 kDa cytokine belonging to the IL-17 family, characterized by a cysteine knot structural motif. Unlike most cysteine knot superfamily members that use three intrachain disulfide bonds, IL-17 family molecules generate the same structural form with only two disulfide links . Mature rat IL-17F consists of 133 amino acids and is typically secreted as a 38 kDa glycosylated disulfide-linked homodimer . Rat IL-17F can also form a 35 kDa disulfide-linked heterodimer with IL-17A, which represents approximately 30% of secreted IL-17F .
Regarding interspecies comparison, mature rat IL-17F shares 59% amino acid sequence identity with human IL-17F and 90% with mouse IL-17F . Within the same species, rat IL-17F shares 55% amino acid identity with rat IL-17A . This high conservation between mouse and rat suggests similar biological functions, while the moderate conservation with human IL-17F indicates potential species-specific differences in activity or regulation that researchers should consider when translating findings between rodent models and human applications.

Which cell types express IL-17F in rats and how is its expression regulated?

In rats, IL-17F is primarily expressed by activated T cells, particularly CD4+ T cells, and activated monocytes . Research has demonstrated that IL-17-expressing T cells also produce IL-17F protein . The expression pattern is notably selective to activated immune cells, suggesting tight regulation of this pro-inflammatory cytokine.
The regulation of IL-17F expression is controlled by the transcription factor RAR-related orphan receptor-γ (RORC), which serves as the key transcriptional regulator for both IL-17A and IL-17F . In experimental models using HLA-B27 transgenic rats, inhibition of RORC significantly suppressed the expression of IL-17F along with IL-17A and IL-22 . This regulatory mechanism appears to be selective, as RORC inhibition did not affect the expression of other T helper cell-related genes .
Expression of IL-17F is typically induced during inflammatory responses, with ex vivo studies showing rapid and selective induction of IL-17F production upon stimulation in disease models . The temporal dynamics of expression show a peak response after approximately 7 days of immunization in certain rat models of inflammation , indicating that timing is a critical factor when designing experiments to study IL-17F expression.

What are the primary biological functions of IL-17F in rats?

IL-17F functions as an effector cytokine of both innate and adaptive immune systems involved in antimicrobial host defense and maintenance of tissue integrity . Its primary roles include:

• Inflammatory regulation: IL-17F is an important regulator of inflammatory responses that appears to function differently than IL-17A in immune responses and diseases . It stimulates the production of various cytokines, chemokines, and adhesion molecules in multiple cell types .

• Neutrophil recruitment: IL-17F plays a significant role in inducing neutrophilia in the lungs and exacerbating antigen-induced pulmonary allergic inflammation . It primarily activates and recruits neutrophils to infection and inflammatory sites .

• Antimicrobial defense: IL-17F stimulates the production of antimicrobial peptides, contributing to host defense against extracellular bacteria and fungi . It induces production of beta-defensins by mucosal epithelial cells, limiting microbial entry through epithelial barriers .

• Cellular proliferation: IL-17F has been shown to stimulate peripheral blood mononuclear cell (PBMC) and T-cell proliferation .

• Angiogenesis inhibition: Interestingly, IL-17F inhibits angiogenesis of endothelial cells and induces endothelial cells to produce IL-2, TGF-beta, and monocyte chemoattractant protein-1 .
  These multifaceted functions highlight IL-17F's complex role in immune regulation and tissue homeostasis in rat models.

What are the most effective methods for detecting and measuring IL-17F expression in rat samples?

Multiple complementary approaches can be employed for robust detection and quantification of IL-17F in rat samples:
For protein detection:

• ELISA: Enzyme-linked immunosorbent assays using specific anti-rat IL-17F antibodies provide quantitative measurement of IL-17F in serum or cell culture supernatants. This approach was effectively used in studies with HLA-B27 transgenic rats to confirm the absence of IL-17F in knockout models and to measure expression levels in various experimental conditions .

• Intracellular cytokine staining: Flow cytometry with specific antibodies like the rat IL-17F antibody (clone 716728) allows for detection of IL-17F at the single-cell level . This technique enables researchers to identify which specific cell populations are producing IL-17F and at what levels .

• Western blotting: For analyzing IL-17F protein in tissue lysates or cellular fractions, western blotting with specific antibodies can detect both monomeric and dimeric forms of IL-17F.
  For gene expression analysis:

• RT-PCR/qPCR: Real-time PCR provides sensitive quantification of IL-17F mRNA levels in various tissues or isolated cell populations. This method was employed in studies examining the effects of RORC inhibition on cytokine expression in rat models .

• RNA sequencing: For genome-wide expression analysis, RNA-seq can be used to examine IL-17F expression in the context of broader transcriptional changes.
  When designing experiments, researchers should consider that IL-17F expression can be transient and cell-type specific. Studies have shown that ex vivo restimulation of splenocytes or lymph node cells might be necessary to detect meaningful levels of IL-17F, with peak responses often occurring within specific time windows after immunization or stimulation .

How can researchers effectively generate and validate IL-17F knockout or transgenic rat models?

Generating and validating IL-17F knockout or transgenic rat models requires careful planning and multifaceted validation approaches:
Generation strategies:

• CRISPR/Cas9 gene editing: Currently the most efficient method for generating IL-17F knockout rats. Design guide RNAs targeting critical exons of the IL-17F gene to create frameshift mutations or large deletions.

• Homologous recombination: For more precise modifications or conditional knockouts, homologous recombination-based approaches can be used, though with lower efficiency than CRISPR.

• Transgenic overexpression: For studying IL-17F overexpression, construct expression vectors with the rat IL-17F coding sequence under control of appropriate promoters (tissue-specific or inducible).
  Validation methods:

• Genomic verification: Perform PCR and sequencing of the targeted locus to confirm the intended genetic modification.

• RNA expression analysis: Use RT-PCR and qPCR to verify the absence (knockout) or increased expression (transgenic) of IL-17F mRNA in relevant tissues .

• Protein validation: Employ ELISA and intracellular cytokine staining to confirm the absence or overexpression of IL-17F protein. Published studies have used these approaches to verify IL-17F knockout models, demonstrating that IL-17F could not be detected in IL-17F KO splenocyte cultures following antigen stimulation .

• Functional validation: Challenge the models with appropriate stimuli known to induce IL-17F production. For instance, immunization with KLH in CFA has been used in IL-17F knockout validation, where subsequent restimulation with KLH should show absence of IL-17F in knockout animals but not in wild-type controls .

• Cross-validation: Assess the expression of related cytokines like IL-17A and IL-22, as alterations in IL-17F can affect their expression patterns. Studies have shown that IL-17F deficiency does not substantially reduce IL-17A expression, while IL-17 knockout leads to reduced IL-17F levels .

What are the optimal conditions for culturing and stimulating rat cells to study IL-17F production?

Optimizing culture and stimulation conditions is crucial for studying IL-17F production in rat cells:
Cell types and isolation:

• Splenocytes: These provide a mixed population containing various IL-17F-producing cells. Isolation through mechanical disruption of the spleen followed by red blood cell lysis yields a suitable population for IL-17F studies .

• Lymph node cells: Cells from draining lymph nodes can be particularly useful in models of localized inflammation or infection .

• Purified T cells: CD4+ T cells can be isolated using magnetic or flow cytometry-based sorting for more focused analysis of T cell-derived IL-17F.
  Culture conditions:

• Medium: RPMI-1640 supplemented with 10% FBS, 2mM L-glutamine, 1mM sodium pyruvate, non-essential amino acids, and antibiotics provides a standard base medium.

• Cell density: 1-2 × 10^6 cells/ml is typically optimal for detecting IL-17F production.
  Stimulation protocols:

• Antigen-specific stimulation: For previously immunized rats, restimulating with the specific antigen (e.g., KLH for KLH-immunized rats) effectively induces IL-17F production . Optimal antigen concentration should be determined empirically.

• T cell receptor stimulation: Anti-CD3/CD28 antibodies (1-5 μg/ml) can be used to activate T cells polyclonally.

• Cytokine polarization: For Th17 differentiation and IL-17F production, supplement cultures with IL-6 (20-50 ng/ml), TGF-β (2-5 ng/ml), IL-23 (20 ng/ml), and anti-IFN-γ antibodies (10 μg/ml).

• Pathogen-associated stimuli: Heat-inactivated Mycobacterium tuberculosis has been used successfully to induce IL-17F in rat models .
  Time course considerations:
  Studies have shown that IL-17F expression follows specific kinetics, with peak production often observed 7 days after immunization in certain models . For in vitro studies, optimal detection times range from 24-72 hours post-stimulation, depending on the specific stimulation method.
  Measurement timing:
  For optimal detection of IL-17F, collect supernatants or harvest cells at intervals between 24-96 hours after stimulation, with peak production often occurring at 48-72 hours for most stimulation protocols.

How does the signaling pathway of IL-17F differ from IL-17A in rat models?

IL-17F and IL-17A signaling in rats share significant similarities but also exhibit important distinctions in receptor utilization, downstream mediators, and biological outcomes:
Receptor engagement:
Both IL-17F and IL-17A signal through receptor complexes involving IL-17RA, but with different configurations. IL-17F signals via IL-17RA-IL-17RC heterodimeric receptor complexes, while it can also signal through IL-17RC homodimeric receptor complexes . IL-17RA-deficient fibroblasts show impaired responses to both IL-17A and IL-17F stimulation, confirming that IL-17RA is required for both IL-17A and IL-17F signaling in vitro and in vivo .
Downstream signaling mechanisms:
The signaling cascade for both cytokines involves:

• Receptor homodimerization or heterodimerization triggers homotypic interaction with TRAF3IP2 (Act1) adapter through SEFIR domains .

• TRAF6-mediated activation of NF-kappa-B and MAP kinase pathways follows, resulting in transcriptional activation of inflammatory genes .

• Both IL-17A and IL-17F signaling are dependent on IL-17RA, Act1, and TRAF6 proteins .
  Functional differences:
  Despite the shared signaling components, IL-17F appears to function differently than IL-17A in immune responses and diseases . These functional differences may arise from:

• Different binding affinities to the receptor complexes

• Recruitment of different auxiliary proteins to the signaling complex

• Cell type-specific responses based on receptor expression patterns
  Unique downstream effects:
  IL-17F homodimers distinctively induce transcriptional activation of IL-33 via IL-17RC, which stimulates group 2 innate lymphoid cells and T-helper 2 cells involved in pulmonary allergic responses to fungi . Additionally, IL-17F promotes sympathetic innervation of peripheral organs by coordinating communication between gamma-delta T cells and parenchymal cells, likely via IL-17RC .
  Heterodimeric signaling:
  IL-17F can form heterodimers with IL-17A , which may have distinct signaling properties and biological activities compared to either homodimer. These heterodimers represent approximately 30% of secreted IL-17F , suggesting they have physiological relevance.

What are the key target genes and proteins regulated by IL-17F in different rat tissues?

IL-17F regulates distinct target genes and proteins across various rat tissues, with some overlap with IL-17A targets but also unique expression patterns:
In epithelial cells and fibroblasts:

• Cytokines: IL-17F stimulates production of IL-6 and CXCL1 in mouse embryonic fibroblasts (MEFs) and peritoneal macrophages . It also induces production of various cytokines in human and likely rat airway epithelial cells, vein endothelial cells, and fibroblasts .

• Chemokines: IL-17F induces expression of multiple chemokines, particularly those involved in neutrophil recruitment, contributing to its role in neutrophilic inflammation .

• Adhesion molecules: IL-17F upregulates adhesion molecules in multiple cell types, facilitating leukocyte recruitment to inflammatory sites .
  In endothelial cells:

• Anti-angiogenic effects: Unlike many inflammatory cytokines, IL-17F inhibits angiogenesis of endothelial cells .

• Secondary mediators: IL-17F induces endothelial cells to produce IL-2, TGF-β, and monocyte chemoattractant protein-1 .
  In immune cells:

• IL-17F stimulates PBMC and T-cell proliferation , suggesting autocrine or paracrine effects on immune cell activation and expansion.
  In respiratory tissues:

• Mucus-related genes: Lung-specific overexpression of IL-17F in mice leads to mucus production , suggesting regulation of mucin genes or pathways controlling goblet cell hyperplasia.

• Inflammatory mediators: IL-17F plays a role in pulmonary recruitment of neutrophils and exacerbation of antigen-induced pulmonary allergic inflammation .
  In joint tissues:

• Matrix turnover mediators: IL-17F regulates cartilage matrix turnover , likely through effects on matrix metalloproteinases and tissue inhibitors of metalloproteinases.
  Tissue-specific effects:
  The expression of IL-17F target genes varies by tissue, with lung-specific overexpression of IL-17F leading to infiltration of lymphocytes and macrophages and mucus production . These tissue-specific effects likely reflect differences in receptor expression and the presence of tissue-specific transcriptional cofactors.

How does IL-17F interact with other cytokines in inflammatory cascades in rat disease models?

IL-17F participates in complex cytokine networks in rat inflammatory models, with significant interactions that shape immune responses:
Synergistic interactions:

• TNF-α and IL-1β: While IL-17F has its own direct effects, studies in MEFs show that TNF-α and IL-1β pathways remain intact in IL-17RA-deficient fibroblasts , suggesting potential for additive or synergistic effects when these pathways are simultaneously activated.

• IL-22: IL-17F expression patterns closely mirror those of IL-22 in inflammatory responses. Levels of IL-22 were moderately reduced in both IL-17F KO and IL-17 KO samples , indicating potential cross-regulation or shared induction pathways.
  Regulatory relationships:

• IL-17A/IL-17F interplay: Levels of IL-17F were reduced in IL-17 KO cells, whereas IL-17F deficiency did not lead to a substantial reduction of IL-17 expression . This suggests asymmetric regulation between these related cytokines.

• RORC-dependent cytokines: In HLA-B27 transgenic rats, RORC inhibition suppressed not only IL-17F but also IL-17A and IL-22 expression , indicating coordinated regulation of these inflammatory cytokines through a common transcription factor.
  Context-dependent interactions:

• Paradoxical effects: In experimental spondyloarthritis models, RORC inhibition suppressed IL-17A, IL-17F, and IL-22 expression but paradoxically accelerated and aggravated disease . This suggests complex interactions with other inflammatory or regulatory pathways that can override the direct effects of IL-17F suppression.

• Disease-specific networks: The role of IL-17F likely varies depending on the disease model. In asthma models, IL-17F was found in airways upon allergen challenge and associated with human asthma and chronic obstructive pulmonary disease , suggesting disease-specific cytokine networks.
  Therapeutic implications:
  The complex interactions observed in experimental models, particularly the paradoxical worsening of spondyloarthritis despite IL-17 axis suppression , highlight the importance of understanding cytokine networks rather than focusing on individual mediators when developing targeted therapies.

What rat models best demonstrate the role of IL-17F in inflammatory diseases?

Several rat models effectively demonstrate IL-17F's role in inflammatory conditions, each offering unique insights:
HLA-B27 transgenic rat model of spondyloarthritis:
This model has been extensively used to study IL-17F in the context of spondyloarthritis (SpA) . Immunization of HLA-B27 transgenic rats with heat-inactivated Mycobacterium tuberculosis induces experimental SpA characterized by both peripheral arthritis and spondylitis . This model shows rapid and selective induction of IL-17A and IL-22 production by various lymphocyte subsets, making it valuable for studying IL-17F's role in SpA pathogenesis .
Airway inflammation models:
Given IL-17F's role in pulmonary neutrophil recruitment and allergic inflammation , models of allergic airway disease are particularly relevant. Adenoviral infection or lipofectamine-mediated gene transfer has been used to acutely overexpress IL-17F in vivo, resulting in pulmonary recruitment of neutrophils . These approaches help understand IL-17F's specific contribution to airway inflammation.
Antigen-induced models:
Immunization with keyhole limpet hemocyanin (KLH) in complete Freund's adjuvant (CFA) has been used to study IL-17F responses . This model allows comparison between wild-type, IL-17F knockout, and IL-17 knockout rats to dissect the specific contributions of these cytokines to immune responses .
Genetic models:
IL-17F knockout rats provide a valuable platform for understanding the non-redundant functions of IL-17F across multiple disease models . Studies with these animals have revealed distinct requirements for IL-17 and IL-17F in different inflammatory responses .
Lung-specific overexpression models:
Transgenic approaches enabling lung-specific overexpression of IL-17F have demonstrated infiltration of lymphocytes and macrophages and mucus production, illustrating IL-17F's tissue-specific inflammatory effects .
Model selection considerations:
When selecting an appropriate model, researchers should consider:

• Disease relevance: Choose models that reflect the human disease of interest

• Temporal dynamics: IL-17F expression shows specific temporal patterns, with peak responses often occurring at distinct timepoints post-stimulation

• Genetic background: The HLA-B27 background provides specific relevance to spondyloarthropathies

• Readout compatibility: Ensure the model allows appropriate assessment of IL-17F-dependent endpoints

How does targeting IL-17F differ from targeting IL-17A in therapeutic approaches for inflammatory conditions in rats?

Targeting IL-17F presents distinct considerations compared to IL-17A-directed approaches in rat models of inflammation:
Mechanistic differences:

• Unique receptor engagement: While both cytokines signal through complexes involving IL-17RA, IL-17F can also signal via IL-17RC homodimeric receptor complexes . This differential receptor utilization suggests selective targeting might yield distinct outcomes.

• Heterodimer considerations: Since IL-17F forms heterodimers with IL-17A (accounting for approximately 30% of secreted IL-17F) , targeting strategies must account for these heterodimers, which may have unique biological activities.
  Functional distinctions:

• Non-redundant roles: Studies with IL-17F and IL-17 knockout rats have revealed distinct requirements for these cytokines in different inflammatory responses , suggesting selective targeting may be preferable for certain conditions.

• Tissue-specific effects: IL-17F plays specific roles in pulmonary inflammation and cartilage homeostasis , which may differ from IL-17A's primary targets.
  Therapeutic implications:

• Paradoxical responses: In HLA-B27 transgenic rat models of spondyloarthritis, RORC inhibition suppressed IL-17A, IL-17F, and IL-22 expression but surprisingly accelerated and aggravated experimental SpA . This paradoxical outcome highlights the complex regulatory networks involving these cytokines and cautions against assumptions about therapeutic outcomes when targeting the IL-17 axis.

• Compensatory mechanisms: The observation that IL-17F levels were reduced in IL-17 KO cells, while IL-17F deficiency did not substantially reduce IL-17 expression , suggests potential compensatory mechanisms that might affect therapeutic outcomes.
  Targeting approaches:

• Direct neutralization: Specific antibodies against IL-17F, such as the rat IL-17F antibody (clone 716728) , provide selective targeting.

• Receptor blockade: Agents targeting IL-17RA affect both IL-17A and IL-17F signaling , while IL-17RC-directed approaches might more selectively impact IL-17F.

• Transcriptional regulation: RORC inhibition affects both IL-17A and IL-17F expression , representing a broader approach to targeting the IL-17 axis.
  The differential outcomes observed with these various approaches underscore the importance of understanding the specific contributions of IL-17F versus IL-17A in different disease contexts to guide rational therapeutic development.

What are the key experimental considerations when assessing IL-17F neutralization in rat inflammatory models?

When evaluating IL-17F neutralization in rat inflammatory models, researchers should address several critical experimental considerations:
Neutralization strategy selection:

• Antibody-based approaches: Using specific anti-IL-17F antibodies like clone 716728 for selective neutralization. Validate antibody specificity and neutralizing capacity in vitro before in vivo applications.

• Receptor blockade: Targeting IL-17RA affects both IL-17A and IL-17F signaling, while IL-17RC-directed approaches might more selectively impact IL-17F .

• Genetic approaches: Utilizing IL-17F knockout rats provides complete elimination of IL-17F but allows developmental compensation .

• Transcriptional inhibition: RORC inhibitors suppress IL-17F along with IL-17A and IL-22 , offering broader IL-17 axis blockade.
  Control considerations:

• Isotype controls: Essential for antibody-based approaches to account for non-specific effects.

• Comparative controls: Include IL-17A neutralization arms to distinguish IL-17F-specific effects from broader IL-17 axis effects.

• Vehicle controls: Particularly important for small molecule inhibitors of RORC or other transcription factors .
  Technical validation:

• Pharmacokinetics/pharmacodynamics: Confirm adequate exposure levels in serum measurements, as demonstrated in RORC inhibitor studies where high compound exposure was verified .

• Target engagement: Verify that IL-17F activity is actually neutralized. For example, confirming inhibition of IL-17F-induced cytokine production in collected tissues or ex vivo functional tests .

• Specificity verification: Ensure that other inflammatory pathways remain intact, as confirmed in studies showing that TNF-α and IL-1β pathways were unaffected in IL-17RA-deficient fibroblasts .
  Outcome assessment:

• Clinical monitoring: Track disease-specific parameters over time, such as arthritis score and hind paw swelling in arthritis models .

• Histopathological analysis: Assess tissue inflammation, destruction, and other relevant parameters at study endpoints .

• Molecular markers: Measure downstream effects on IL-17F target genes and proteins across relevant tissues .

• Unexpected outcomes: Be prepared for paradoxical results, as seen in the HLA-B27 transgenic rat model where RORC inhibition worsened rather than improved disease despite suppressing IL-17 axis cytokines .
  Timing considerations:

• Prophylactic versus therapeutic: Design studies to evaluate both preventive and treatment effects of IL-17F neutralization, as outcomes may differ substantially .

• Duration of intervention: Consider both acute and chronic neutralization, as compensatory mechanisms may emerge over time.

• Assessment timing: Plan sample collection based on known expression kinetics of IL-17F in the specific model being used .

How do heterodimers of IL-17A/IL-17F differ functionally from homodimers in rat inflammatory models?

The functional distinctions between IL-17A/F heterodimers and their respective homodimers represent an important but understudied area in rat inflammatory models:
Structural and expression considerations:
IL-17F can form both 38 kDa glycosylated disulfide-linked homodimers and 35 kDa disulfide-linked heterodimers with IL-17A . These heterodimers account for approximately 30% of secreted IL-17F , suggesting they have physiological relevance rather than being merely incidental.
Receptor utilization differences:

What paradoxical findings have been observed in IL-17F research in rats and how can these be reconciled?

Several paradoxical findings in rat IL-17F research challenge straightforward interpretations of this cytokine's role:
RORC inhibition paradox:
The most striking paradoxical finding comes from studies with HLA-B27 transgenic rats, where RORC inhibition suppressed IL-17A, IL-17F, and IL-22 expression but unexpectedly accelerated and aggravated experimental spondyloarthritis . This treatment significantly accelerated the onset of both spondylitis and arthritis and increased disease severity despite effective suppression of the IL-17 axis . Histopathological analysis confirmed increased inflammatory infiltration, destruction, new bone formation, and hypertrophic chondrocytes in RORC inhibitor-treated rats .
Possible explanations:

• Compensatory inflammation: Suppression of the IL-17 axis might upregulate alternative inflammatory pathways that drive more severe disease.

• Regulatory functions: IL-17F might have unrecognized regulatory functions in certain contexts, similar to the dual pro-inflammatory and regulatory roles described for cytokines like IFN-γ.

• Cell-specific effects: RORC inhibition affects multiple cell types beyond Th17 cells, potentially disrupting regulatory populations.

• Temporal considerations: The timing of IL-17F activity might determine whether it has protective or pathogenic effects.

• Gut-joint axis disruption: Despite no obvious gut inflammation being observed following RORC inhibition , subtle alterations in gut immunity might contribute to joint disease exacerbation.
  IL-17F versus IL-17A genetic deletion effects:
  Another paradoxical observation is that IL-17F levels were reduced in IL-17 KO cells, whereas IL-17F deficiency did not substantially reduce IL-17 expression . This asymmetric relationship suggests complex regulatory mechanisms between these closely related cytokines.
  Reconciliation approaches:

• Context-dependent analyses: Examine IL-17F's role across different tissues, disease stages, and inflammatory contexts to map out when it promotes versus constrains inflammation.

• Comprehensive immune profiling: When paradoxical outcomes occur, broad immune profiling might reveal compensatory mechanisms or unexpected cellular shifts.

• Combinatorial targeting: Test combination approaches that target IL-17F alongside other inflammatory or regulatory pathways to better understand the cytokine network dynamics.

• Temporal modulation: Investigate whether timed or pulsed inhibition of IL-17F might yield different outcomes than continuous suppression. The paradoxical findings highlight that cytokine biology is rarely straightforward, and that IL-17F likely functions within complex networks where its role can vary dramatically depending on context. These observations underscore the importance of thorough preclinical evaluation before translating IL-17F-targeted therapies to clinical applications.