1. Bibliographic Information

1.1. Title

Lack of TYK2 signaling enhances host resistance to Candida albicans skin infection.

1.2. Authors

Sara Miranda, Caroline Lassnig, Kristina Schmidhofer, Hrönn Kjartansdottir, Claus Vogl, Simone Tangermann, Irina Tsymala, Verena Babl, Mathias Müller, Karl Kuchler & Birgit Strobl.

The authors are affiliated with several institutions, primarily the University of Veterinary Medicine Vienna and the Medical University of Vienna, Austria. Their collective expertise spans immunology, molecular biology, genetics, and infectious diseases, which is well-suited for this study's focus on host-pathogen interactions at the molecular level.

1.3. Journal/Conference

The paper was published in Nature Communications. This is a high-impact, peer-reviewed, open-access scientific journal published by Springer Nature. It is highly reputable and covers a wide range of fields in the natural sciences, including biology, physics, chemistry, and earth sciences. Publication in this journal signifies that the work is considered to be of high quality and significant importance.

1.4. Publication Year

2024 (Published online: 03 December 2024).

1.5. Abstract

The abstract summarizes the study's investigation into the role of Tyrosine kinase 2 (TYK2) in the immune response to skin infections caused by Candida albicans, the most common human fungal pathogen. While TYK2 is typically known for its protective role against various microbes, this study reveals a surprising, detrimental function in fungal skin infections. The authors found that mice lacking TYK2 or its enzymatic (kinase) activity showed enhanced resistance to C. albicans skin infections. This resistance was characterized by limited fungal spread and faster wound healing. Mechanistically, the absence of TYK2 signaling led to the formation of a unique layer of necrotic neutrophils surrounding the fungus. Transcriptomic analysis of neutrophils revealed that TYK2 regulates interferon-inducible genes, affecting the neutrophils' antifungal capabilities. Furthermore, the study demonstrates that TYK2-dependent production of interferon-gamma ($IFNγ$) contributes to the spread (dissemination) of the fungus from the skin to the kidneys. The paper concludes that TYK2 plays a previously unrecognized harmful role in cutaneous candidiasis.

1.6. Original Source Link

The original paper can be accessed via the provided link: /files/papers/691ac7db110b75dcc59ae425/paper.pdf. The paper is officially published.

---

2. Executive Summary

2.1. Background & Motivation

Candida albicans is a fungus that commonly lives harmlessly on human skin and mucous membranes. However, in individuals with weakened immune systems or compromised skin barriers, it can cause infections ranging from superficial skin rashes to life-threatening systemic disease. The immune system must therefore maintain a delicate balance: tolerating the harmless commensal form while effectively fighting off invasive infections.

A key player in the immune system is Tyrosine kinase 2 (TYK2), a protein that helps transmit signals from various communication molecules called cytokines, which orchestrate immune responses. Previous research has established that TYK2 is crucial for defending against many bacterial and viral infections. Paradoxically, overactive TYK2 signaling is also implicated in autoimmune diseases like psoriasis, leading to the development of drugs that inhibit TYK2 (e.g., Deucravacitinib).

This created a critical knowledge gap: What is the specific role of TYK2 in the immune response to fungal infections, particularly at barrier tissues like the skin? Understanding this is vital, as TYK2-inhibiting drugs could inadvertently affect a patient's ability to fight off common fungal pathogens. The study was motivated by this lack of understanding and aimed to dissect the precise function of TYK2—including both its enzymatic (kinase) and non-enzymatic (scaffolding) roles—during a cutaneous C. albicans infection.

2.2. Main Contributions / Findings

The paper's primary finding is a paradigm shift: it reveals that TYK2 signaling is detrimental to the host during a C. albicans skin infection, which is in stark contrast to its known protective roles against other pathogens.

The key contributions are:

1. Enhanced Host Resistance in the Absence of TYK2: The study conclusively shows that mice completely lacking TYK2 ($Tyk2⁻/⁻$) or expressing a version without enzymatic activity ($Tyk2^K923E$) are more resistant to cutaneous C. albicans infection. They exhibit lower fungal burdens in the skin, significantly reduced spread to the kidneys, and faster wound healing compared to normal (wild-type) mice.

2. Identification of a Novel Defense Mechanism: The authors discovered that in TYK2-deficient mice, neutrophils (a type of white blood cell) form a distinct, dense layer of necrotic (dead) cells that physically walls off the invading fungus. This "necrotic barrier" appears to contain the infection locally and prevent it from spreading deeper into the tissues and bloodstream.

3. **Elucidation of the TYK2-` IFNγ$Axis:** The study pinpoints the molecular pathway responsible for this effect. `TYK2` is required for the production of a specific cytokine, **interferon-gamma ($IFNγ$)**, at the infection site. By showing that mice unable to respond to$IFNγ$($Ifngr1⁻/⁻$) phenocopy the `TYK2`-deficient mice (i.e., they are also resistant), the authors demonstrate that `TYK2` exerts its harmful effect by promoting$IFNγ$production. This$IFNγ$ signal appears to prevent the formation of the protective necrotic neutrophil layer.

4. Pinpointing the Cellular Source of $IFNγ$: Through a series of elegant experiments using different genetically modified mice, the study identifies $γδ$ T cells (a type of "innate-like" lymphocyte prevalent in the skin) as the primary source of the detrimental, TYK2-dependent $IFNγ$ during the infection.

   In summary, this research uncovers a novel and unexpected immunoregulatory pathway where TYK2 signaling, via $IFNγ$ produced by $γδ$ T cells, undermines the host's ability to contain a fungal skin infection.

---

3. Prerequisite Knowledge & Related Work

3.1. Foundational Concepts

• Candida albicans: A species of yeast that is one of the most common fungal pathogens in humans. It is a part of the normal microflora in the mouth, gut, and skin. However, it is an opportunistic pathogen, meaning it can cause infections (called candidiasis) when the host's immune system is weakened or when the normal balance of microflora is disrupted (e.g., by antibiotics). Infections can be superficial (e.g., oral thrush, skin rashes) or invasive and life-threatening if the fungus enters the bloodstream.

• Innate and Adaptive Immunity: The immune system has two main branches.

  • Innate Immunity: The body's first line of defense. It is non-specific and provides a rapid response to pathogens. Key cells include neutrophils (phagocytes that engulf and kill microbes), macrophages (large phagocytic cells), and dendritic cells.
  • Adaptive Immunity: A more specialized defense that develops over time and provides long-lasting memory. Key cells are lymphocytes, such as T cells (which coordinate the immune response and kill infected cells) and B cells (which produce antibodies).

• Cytokines: Small proteins that act as messengers between cells, playing a critical role in regulating immune responses. Key cytokines in this paper include:

  • Interferons (IFNs): A group of cytokines originally named for their ability to "interfere" with viral replication.
    • $Type I IFNs (IFN-α/β)$: Primarily involved in antiviral defense.
    • $Type II IFN (IFNγ)$: A pro-inflammatory cytokine crucial for activating macrophages and coordinating defense against intracellular pathogens.
  • Interleukins (ILs): A large group of cytokines that mediate communication between white blood cells. IL-12 and IL-23 are important for T cell activation and differentiation.

• Janus Kinase (JAK)-STAT Pathway: A primary signaling pathway used by many cytokines to transmit signals from the cell surface to the nucleus, where they alter gene expression.

  1. A cytokine binds to its specific receptor on the cell surface.
  2. This binding brings together Janus kinases (JAKs) associated with the receptor.
  3. The JAKs phosphorylate (add a phosphate group to) each other and the receptor, activating them.
  4. This creates a docking site for proteins called STATs (Signal Transducers and Activators of Transcription).
  5. The JAKs phosphorylate the STATs.
  6. The phosphorylated STATs form pairs (dimers), move into the nucleus, and bind to DNA to turn specific genes on or off.

• Tyrosine Kinase 2 (TYK2): TYK2 is a member of the JAK family. It is a non-receptor tyrosine kinase, meaning it adds phosphate groups to tyrosine residues on other proteins. It has two key functions:

  • Kinase (Enzymatic) Activity: The ability to phosphorylate other proteins, which is essential for signal transduction in the JAK-STAT pathway.
  • Scaffolding Function: A structural role in stabilizing cytokine receptors on the cell surface, which is independent of its kinase activity.

• Genetically Modified Mouse Models: The study uses several mouse models to dissect the function of TYK2.

  • $Tyk2⁻/⁻$ (Knockout): Mice in which the Tyk2 gene has been completely deleted. They lack the TYK2 protein entirely.
  • $Tyk2^K923E$ (Kinase-Inactive): Mice that express a mutated TYK2 protein where a single amino acid change renders its kinase function inactive. This model allows researchers to separate the kinase-dependent functions from the scaffolding functions.
  • $Ifngr1⁻/⁻$ (Receptor Knockout): Mice lacking the receptor for $IFNγ$. These mice cannot respond to $IFNγ$ signals.
  • $Rag2⁻/⁻$: Mice lacking the RAG2 gene, which is essential for the development of mature T and B cells. These mice have a severely compromised adaptive immune system.
  • $Tyk2^ΔT$ (Conditional Knockout): Mice where the Tyk2 gene is specifically deleted only in certain T cell populations ($αβ$ T cells and NKT cells) using the CD4-Cre system.

3.2. Previous Works

The authors build upon a well-established but seemingly contradictory body of literature on TYK2.

• Protective Role of TYK2: Numerous studies have shown that TYK2 is essential for host defense. Humans and mice lacking TYK2 are highly susceptible to various bacterial, viral, and mycobacterial infections (e.g., tuberculosis). This is largely attributed to impaired signaling from Type I IFNs and IL-12, which leads to deficient $IFNγ$ production by T cells and NK cells.
• Pathogenic Role of TYK2: In contrast, genetic studies in humans have linked variations in the TYK2 gene to an increased risk of numerous autoimmune and inflammatory diseases, such as psoriasis, Crohn's disease, and multiple sclerosis. In these conditions, TYK2-mediated signaling (e.g., from IL-23) promotes chronic inflammation. This has made TYK2 a prime target for therapeutic inhibition.
• Immunity to Cutaneous Candidiasis: Previous research on skin immunity to C. albicans has heavily focused on the IL-17/IL-23 axis. Cytokines like IL-23 stimulate various immune cells, particularly $γδ$ T cells, to produce IL-17. IL-17 is critical for recruiting neutrophils to the site of infection and is generally considered protective against fungal infections at mucosal and skin surfaces. The role of $IFNγ$ in this context has been less clear and sometimes controversial.

3.3. Technological Evolution

The study of TYK2 and other JAKs has evolved significantly. Initially, research relied on complete knockout mice ($Tyk2⁻/⁻$), which showed the overall importance of the protein but couldn't distinguish between its different functions. The development of more sophisticated genetic tools has been crucial:

• Kinase-Inactive Models ($Tyk2^K923E$): These models were a major step forward, allowing scientists to separate the catalytic (signaling) function of TYK2 from its structural (scaffolding) role.
• Conditional Knockout Systems (Cre-Lox): These systems enable the deletion of a gene in specific cell types or at specific times, providing much finer control and allowing researchers to pinpoint which cells are responsible for a particular effect (as seen in the $Tyk2^ΔT$ mice).
• Transcriptomics (RNA-sequencing): This technology allows for an unbiased, genome-wide analysis of gene expression in specific cell populations (like neutrophils sorted from the infection site), providing deep mechanistic insights that were previously unattainable.
• Selective Inhibitors: The clinical development of drugs like Deucravacitinib, which specifically inhibit TYK2, has brought the basic science of TYK2 function into sharp clinical focus, driving the need for studies like this one to understand the full immunological consequences of TYK2 inhibition.

3.4. Differentiation Analysis

This paper's core innovation lies in its discovery of a context-dependent, detrimental role for TYK2.

• Contrast with Established Dogma: While most prior work emphasized TYK2's protective function in immunity, this study demonstrates the opposite in the context of a specific pathogen (C. albicans) at a specific site (the skin). This challenges the simple view of TYK2 as a universally "good" immune component.

• Kinase Activity is Key: By showing that both $Tyk2⁻/⁻$ and $Tyk2^K923E$ mice have the same enhanced resistance, the study pinpoints the kinase activity of TYK2 as the driver of the detrimental effect. Its scaffolding function is not involved in this particular pathology.

• Novel Mechanism of Fungal Containment: The paper proposes a novel defense mechanism: the formation of a necrotic neutrophil barrier. While neutrophil recruitment is known to be important, the idea that a layer of dead neutrophils can effectively wall off and contain the fungus is a new and intriguing concept.

• Systematic Dissection of the Immune Pathway: Compared to studies that might focus on a single cytokine or cell type, this work stands out for its methodical approach. It uses a series of different genetic mouse models to trace the pathogenic signal from the intracellular molecule (TYK2) to the systemic cytokine ($IFNγ$), and finally to the specific cellular source ($γδ$ T cells), providing a complete and compelling story.

  ---

4. Methodology

The study employs a multi-faceted approach centered on genetically modified mouse models to investigate the role of TYK2 in vivo. The methodology can be broken down into a series of logical experimental steps.

4.1. Animal Models and Infection Protocol

• Principles: The core idea is to compare the immune response of normal (Wild-Type, WT) mice to that of mice with specific genetic modifications in the TYK2-` IFNγ$ signaling pathway.
• Core Methodology:
  1. Mouse Strains: A panel of mouse strains on a C57BL/6 background was used: WT, $Tyk2⁻/⁻$ (total knockout), $Tyk2^K923E$ (kinase-inactive), $Ifngr1⁻/⁻$ ($IFNγ$ receptor knockout), $Rag2⁻/⁻$ (no T/B cells), and $Tyk2^ΔT$ (Tyk2 deleted in $αβ$ T and NKT cells). Using age- and sex-matched mice minimizes variability.
  2. Pathogen: A well-characterized strain of C. albicans (SC5314) was grown to a logarithmic phase.
  3. Infection: Mice were anesthetized, and their back skin was shaved. They were then injected intradermally (into the skin) with a high dose of C. albicans ($1 \times 10^8$ Colony-Forming Units, or CFU). This model mimics a deep skin wound infection that can potentially become systemic. Control mice were injected with PBS (a saline solution).

4.2. Assessment of Disease Progression and Fungal Burden

• Principles: To quantify the severity of the infection and determine if the host is successfully controlling the pathogen.
• Core Methodology:
  1. Fungal Load (CFU Assay): At specific time points (day 2 and 4 post-infection), skin biopsies and kidneys were harvested. The tissues were weighed, homogenized, and serially diluted. The dilutions were plated on YPD-agar plates, which support fungal growth. After incubation, the number of C. albicans colonies was counted. The result is expressed as CFU per gram of tissue, providing a direct measure of the live fungal burden.
  2. Clinical Monitoring: Mice were monitored for up to 24 days. Wound healing was visually assessed (disappearance of nodules, ulcers, and crusts), and body weight was recorded as a general indicator of health.

4.3. Immunological and Histological Analysis

• Principles: To visualize the location of the fungus and immune cells within the tissue and to quantify the immune cell populations recruited to the site of infection.
• Core Methodology:
  1. Histology: Skin biopsies were fixed in formaldehyde and embedded in paraffin. Thin sections were cut and stained.
     • Gomori Methenamine-Silver (GMS) Stain: A specific stain that colors fungi black, allowing visualization of their location and invasion depth in the skin tissue.
     • Immunohistochemistry (IHC): Uses antibodies to detect specific proteins in the tissue sections. Antibodies used were:
       • NIMP-R14: Stains neutrophils and inflammatory monocytes.
       • Ki-67: A marker for cellular proliferation. In this context, Ki-67-positive neutrophils are considered activated, while Ki-67-negative cells are either quiescent or dead.
       • MPO (Myeloperoxidase): An enzyme abundant in activated neutrophils.
  2. Flow Cytometry: To analyze the cellular composition of the infected skin. Skin tissue was digested with enzymes to create a single-cell suspension. These cells were then stained with fluorescently-labeled antibodies against various cell surface markers (e.g., CD45 for all immune cells, CD11b for myeloid cells, Ly6G for neutrophils, Ly6C for monocytes, $TCRβ$ for $αβ$ T cells, $TCRγδ$ for $γδ$ T cells). A flow cytometer then analyzes thousands of individual cells, allowing for precise quantification of different immune cell populations.
  3. Cytokine Measurement:
     • RT-qPCR (Reverse Transcription Quantitative PCR): Measures the amount of specific messenger RNA (mRNA) to quantify gene expression. RNA was isolated from skin biopsies, converted to cDNA, and the levels of Ifng mRNA and other genes were measured.
     • Luminex Assay: A bead-based immunoassay used to measure the protein levels of multiple cytokines ($IFNγ$, IL-1β, IL-23) simultaneously in skin homogenates.

4.4. Functional and Mechanistic Studies

• Principles: To test causal relationships and dissect the molecular pathways involved.
• Core Methodology:

  1. Neutrophil Depletion: To test if neutrophils were responsible for the enhanced protection in $Tyk2⁻/⁻$ mice, mice were injected with an anti-Ly6G antibody, which specifically binds to and eliminates neutrophils from the body. The effect of this depletion on fungal dissemination to the kidneys was then measured.

  2. Transcriptomics (RNA-sequencing): To get an unbiased view of how TYK2 affects neutrophil function. Myeloid cells (mostly neutrophils) were sorted from the infected skin of WT, $Tyk2⁻/⁻$, and $Tyk2^K923E$ mice using FACS (Fluorescence-Activated Cell Sorting). The RNA from these purified cells was then sequenced to identify all genes that were differentially expressed between the groups. This revealed the downstream effects of TYK2 signaling on the neutrophil transcriptome.

  3. In Vitro T Cell Stimulation: To determine if $γδ$ T cells could be a source of $IFNγ$. Single-cell suspensions from the skin or spleen of WT and $Tyk2⁻/⁻$ mice were cultured in vitro and stimulated with either IL-12 (a known $IFNγ$ inducer) or heat-killed C. albicans. The production of $IFNγ$ by $γδ$ T cells was then measured using intracellular flow cytometry.

     ---

5. Experimental Setup

5.1. Datasets

This study did not use pre-existing public datasets. Instead, it generated its own data through controlled laboratory experiments.

• Biological "Dataset":
  • Pathogen: Candida albicans strain SC5314, a widely used and well-characterized clinical isolate.
  • Host: Several lines of genetically engineered mice, all on the $C57BL/6N$ genetic background to ensure comparability. The primary comparison group (baseline) is the wild-type $C57BL/6N$ mouse.
• Choice Rationale: The use of a standardized fungal strain and isogenic mouse lines (mice that are genetically identical except for the gene of interest) is crucial for creating a controlled experimental system. This setup allows the researchers to attribute any observed differences in immune response and disease outcome directly to the genetic modification (e.g., the absence of TYK2).

5.2. Evaluation Metrics

The study uses several quantitative metrics to evaluate the outcomes of the infection.

• Colony-Forming Unit (CFU):

  • Conceptual Definition: CFU is a measure used to estimate the number of viable (live and capable of reproducing) bacteria or fungi in a sample. The principle is that a single viable fungal cell, when placed on a nutrient-rich medium, will grow and divide to form a visible colony. By counting the colonies, one can infer the number of viable cells in the original sample. It is a fundamental metric for quantifying microbial load in infected tissues.
  • Calculation: The result is typically expressed as CFU per gram of tissue. While there isn't a single formula, the calculation process is: $ \text{CFU/g} = \frac{\text{(Number of colonies)} \times \text{(Dilution factor)}}{\text{Volume of culture plated (in mL)}} \times \frac{\text{Total volume of homogenate (in mL)}}{\text{Weight of tissue (in g)}} $
  • Symbol Explanation:
    • Number of colonies: The average count of colonies on the agar plate.
    • Dilution factor: The reciprocal of the dilution used for plating (e.g., if a 1:1000 dilution is used, the factor is 1000).
    • Volume of culture plated: The volume of the diluted sample spread on the plate.
    • Total volume of homogenate: The total liquid volume the tissue was homogenized in.
    • Weight of tissue: The initial weight of the tissue sample.

• Statistical Significance (p-value):

  • Conceptual Definition: The p-value represents the probability of observing the experimental results (or more extreme results) if there were actually no real effect or difference between the groups being compared (the "null hypothesis"). A small p-value (typically < 0.05) indicates that the observed result is unlikely to be due to random chance, leading to the conclusion that there is a statistically significant difference between the groups.
  • Tests Used:
    • One-way ANOVA (Analysis of Variance) with Tukey's multiple comparison test: Used to compare the means of three or more groups (e.g., WT vs. $Tyk2⁻/⁻$ vs. $Tyk2^K923E$). ANOVA determines if there is an overall difference among the groups, and Tukey's test then identifies which specific pairs of groups are significantly different from each other.
    • Two-tailed Mann-Whitney test: A non-parametric test used to compare two independent groups when the data does not follow a normal distribution (common for CFU data).
    • Log-rank (Mantel-Cox) test: Used to compare survival curves or time-to-event curves (in this case, the wound healing curves in Figure 1g). It tests the null hypothesis that there is no difference in the probability of an event (healing) occurring between the groups over time.

5.3. Baselines

• Primary Baseline: The wild-type (WT) C57BL/6N mouse is the main control group in almost all experiments. The immune response and disease progression in all genetically modified mice are compared against this standard to determine the effect of the specific gene alteration.
• Experimental Controls:

  • PBS-injected mice: In many experiments, a group of mice is injected with Phosphate-Buffered Saline (PBS) instead of C. albicans. This serves as a negative control to establish the baseline state of the tissue and immune system in the absence of infection.

  • Internal Controls in Genetic Crosses: When comparing, for example, $Tyk2^ΔT$ mice, the proper control group is the $Tyk2^fl/fl$ littermate that does not carry the CD4-Cre transgene. This controls for any potential effects of the "floxed" allele itself.

    These baselines are representative and appropriate because they allow for the direct and controlled assessment of the function of the target gene (Tyk2, Ifngr1, Rag2) in the specific context of the C. albicans skin infection model.

---

6. Results & Analysis

This section deconstructs the key findings of the paper, following the logical flow of the experiments presented in its figures.

6.1. Core Results Analysis

6.1.1. Absence of TYK2 or its Kinase Activity Enhances Resistance to Cutaneous Candidiasis (Figure 1)

The study first established the core phenotype. Mice lacking TYK2 ($Tyk2⁻/⁻$) or its kinase activity ($Tyk2^K923E$) were infected intradermally with C. albicans.

• Fungal Burden: Both $Tyk2⁻/⁻$ and $Tyk2^K923E$ mice showed significantly lower fungal loads in the skin compared to WT mice at day 2 and day 4 post-infection (p.i.). More strikingly, while WT mice had substantial fungal dissemination to the kidneys, the TYK2-mutant mice had almost no detectable fungus in their kidneys. This demonstrates that TYK2's kinase activity is required for both local fungal proliferation and systemic spread.

• Systemic Immune Response: Infection in WT mice caused a significant increase in granulocytes (neutrophils) in the blood, a typical sign of systemic inflammation. This response was much less pronounced in TYK2-mutant mice, consistent with a more contained, local infection.

• Fungal Containment and Healing: GMS staining of skin sections showed that in WT mice, fungal hyphae spread throughout the skin layers. In contrast, in TYK2-mutant mice, the fungi remained largely confined to the initial infection site. Correspondingly, $Tyk2⁻/⁻$ and $Tyk2^K923E$ mice healed their wounds significantly faster than WT mice.

  The following figure (Figure 1 from the original paper) presents the data supporting these conclusions.

  该图像是图表，展示了不同小鼠模型在皮肤和肾脏感染后 C. albicans 的真菌负荷。图中包含对比不同基因型小鼠在感染后 2 天和 4 天的真菌计数（CFU/g 组织），并分析了血细胞组成及存活率。

6.1.2. A Necrotic Neutrophil Layer Forms in TYK2-Mutant Mice (Figure 2)

The researchers next investigated the immune cell infiltrate in the skin to understand the mechanism of enhanced resistance.

• Neutrophil Recruitment is Unaffected: Using IHC and flow cytometry, the study found that the recruitment of neutrophils and monocytes to the infection site was similar across all genotypes (WT, $Tyk2⁻/⁻$, and $Tyk2^K923E$). This crucial finding indicates that TYK2 does not affect the initial alarm signals that call immune cells to the skin.

• Formation of a Ki-67-Negative Layer: A striking difference was observed with Ki-67 staining. Ki-67 is a marker of active, proliferating cells. In WT mice, neutrophils surrounding the fungi were largely Ki-67-positive. However, in $Tyk2⁻/⁻$ and $Tyk2^K923E$ mice, a distinct layer of Ki-67-negative cells formed immediately adjacent to the fungal mass by day 2 p.i. Pathological examination confirmed these cells showed signs of necrosis (unprogrammed cell death). This suggests that in the absence of TYK2 signaling, neutrophils undergo necrosis more readily, forming a physical barrier that may trap the pathogen.

  The following figure (Figure 2 from the original paper) shows the histological evidence for this necrotic neutrophil layer.

  该图像是图表，展示了缺乏TYK2的小鼠在Candida albicans皮肤感染后的中性粒细胞反应。图中包括B（血液）和C（皮肤）处的中性粒细胞分布分析，以及D显示的组织切片结果，表明TYK2缺失对中性粒细胞活性的影响。

6.1.3. Neutrophils are Essential for the Enhanced Fungal Containment (Figure 3)

To test if this neutrophil barrier was causally linked to the reduced fungal dissemination, the researchers performed a depletion experiment.

• Depletion Abolishes Protection: WT and $Tyk2⁻/⁻$ mice were treated with an anti-Ly6G antibody to eliminate neutrophils. In these neutrophil-depleted mice, the difference in fungal dissemination between genotypes disappeared. $Tyk2⁻/⁻$ mice now had high levels of fungus in their kidneys, comparable to WT mice. GMS staining also showed increased fungal penetration into deeper skin layers in both genotypes after depletion.

• Conclusion: This experiment provides strong evidence that neutrophils are the critical cell type responsible for the enhanced resistance seen in $Tyk2⁻/⁻$ mice. The necrotic barrier they form is functionally important for preventing systemic spread.

  The following figures (Figures 3, 4, 5 from the original paper) illustrate the experimental setup and results of the neutrophil depletion study.

  该图像是图表，展示了在PBS和抗Ly6G处理下，WT和Tyk2^-/-小鼠感染C. albicans后的组织切片。不同处理组的切片显示出不同的组织结构变化，且分别标示了每个组别。图中包含病理学染色结果，标尺显示数据精度。

  该图像是一个柱状图，展示了不同处理下小鼠肾脏中Candida albicans的菌落形成单位（CFU）。横坐标为处理组（PBS和anti-Ly6G），纵坐标为CFU/g组织，显示Tyk2缺失小鼠（红色）在抗真菌能力上与野生型小鼠（灰色）存在显著差异，P值均小于0.0001。

  该图像是图表，展示了缺乏TYK2或其激酶活性对皮肤浸润的髓系细胞转录组的影响。包括火山图展示了与野生型（WT）小鼠相比，$T y k 2^{ ext{'}}, T y k 2^{ ext{K 9 2 3 E}}$小鼠的差异表达基因，以及热图显示的差异基因表达情况，强调了干扰素刺激基因（ISGs）。

6.1.4. TYK2 Regulates Interferon-Stimulated Gene Expression in Neutrophils (Figure 4)

To understand how TYK2 alters neutrophil function, the team performed RNA-sequencing on myeloid cells (mostly neutrophils) sorted from the infected skin.

• Transcriptomic Profile: A large number of genes were differentially expressed between WT and TYK2-mutant neutrophils. The most significantly downregulated genes in $Tyk2⁻/⁻$ and $Tyk2^K923E$ neutrophils were interferon-stimulated genes (ISGs). Pathway analysis confirmed a strong defect in pathways related to "interferon alpha/beta signaling" and "interferon signaling."

• $IFNγ$ Production is TYK2-Dependent: The researchers then measured cytokine levels at the infection site. They found that the production of $IFNγ$, both at the mRNA and protein level, was strongly induced upon infection in WT mice but was severely impaired in $Tyk2⁻/⁻$ and $Tyk2^K923E$ mice.

• Connecting the Dots: These results link the absence of TYK2 kinase activity to a defective interferon response in skin-infiltrating neutrophils and reduced local $IFNγ$ production.

  The following figure (Figure 4 from the original paper) shows the transcriptomic data and $IFNγ$ measurements.

  该图像是图表，展示了Ifngr1/小鼠与WT小鼠在皮肤和肾脏中对C. albicans感染的敏感性。图中比较了两组小鼠的真菌负荷（b、c），血细胞组成（d），以及感染后皮肤组织的GMS染色（e）和Ki-67抗体染色（f）。

6.1.5. IFNγ Signaling is Detrimental and Phenocopies the TYK2 Effect (Figure 5)

The previous result suggested that $IFNγ$ might be the detrimental factor promoted by TYK2. To test this directly, they used mice lacking the $IFNγ$ receptor ($Ifngr1⁻/⁻$).

• $Ifngr1⁻/⁻$ Mice are Resistant: $Ifngr1⁻/⁻$ mice, when infected with C. albicans, showed the same phenotype as TYK2-mutant mice: they had comparable fungal load in the skin but significantly reduced fungal dissemination to the kidneys.

• Formation of the Necrotic Layer: Crucially, $Ifngr1⁻/⁻$ mice also formed the characteristic Ki-67-negative necrotic cell layer around the fungus, just like the TYK2 mutants.

• Conclusion: This experiment provides powerful evidence that TYK2 exerts its detrimental effect by promoting $IFNγ$ signaling. It is the $IFNγ$ itself that prevents the formation of the protective necrotic barrier and facilitates fungal spread.

  The following figure (Figure 5 from the original paper) demonstrates the phenocopy between TYK2-mutant and $Ifngr1⁻/⁻$ mice.

  该图像是图表，展示了RAG2缺乏对C. albicans组织扩散及IFNγ生成的影响。图a显示了实验流程，图b展示了皮肤中Ifng的mRNA水平，图c和d分别展示了皮肤及肾脏的CFU计数，图e是GMS染色的皮肤切片，图f展示了PBS和感染后4天的细胞群体分布。

6.1.6. T/B Cells, Not NK Cells, are the Source of Pathogenic IFNγ (Figures 6-8)

The final piece of the puzzle was to identify which immune cell type was producing the harmful, TYK2-dependent $IFNγ$.

• Role of Adaptive Immunity ($Rag2⁻/⁻$ mice): $Rag2⁻/⁻$ mice, which lack mature T and B cells, were infected. These mice failed to produce $IFNγ$ in the skin and showed profoundly reduced fungal dissemination to the kidneys, similar to TYK2-mutant mice. This result implicates T cells (or possibly B cells) as the critical source of $IFNγ$.

• Excluding $αβ$ T cells and NKT cells ($Tyk2^ΔT$ mice): Next, they used $Tyk2^ΔT$ mice, where TYK2 is deleted specifically in $αβ$ T cells and NKT cells. These mice were not protected; they had high fungal loads in the kidneys and normal $IFNγ$ production, just like WT mice. This rules out $αβ$ T cells and NKT cells as the source of the TYK2-driven detrimental $IFNγ$.

• Identifying $γδ$ T cells: By elimination, $γδ$ T cells were the prime suspects. To test this, skin cells from WT and $Tyk2⁻/⁻$ mice were stimulated in vitro with heat-killed C. albicans. The results were clear: WT $γδ$ T cells produced robust amounts of $IFNγ$, but $Tyk2⁻/⁻$ $γδ$ T cells produced very little. This demonstrates that $γδ$ T cells are capable of producing $IFNγ$ in response to C. albicans in a TYK2-dependent manner.

  Together, these results build a compelling case that innate-like $γδ$ T cells are the primary source of the pathogenic $IFNγ$ that drives fungal dissemination in this model.

The following figures (Figures 6, 7, and 8 from the original paper) detail the experiments used to pinpoint the cellular source of $IFNγ$.

该图像是一个示意图，展示了缺乏TYK2信号的鼠模型在皮肤和肾脏中对C. albicans感染的抵抗力。图中显示了不同细胞类型的分布情况，包括粒细胞和淋巴细胞，同时展示了转录组分析中Ifng和IFNγ的表达情况。该研究揭示TYK2在真菌感染中的重要作用。

该图像是图表，展示了不同基因型小鼠（WT 和 TYK2−/−）的 γδ T 细胞在 C. albicans 刺激下的反应。图中包括了单细胞悬液的提取、培养和流式细胞术分析的结果。面板 b 显示了不同刺激条件（IL-2、IL-12 和 HK C.a）下，IFNγ 产生的 γδ T 细胞的百分比（c）和数量（d）。统计分析结果表明，TYK2 缺失显著影响了免疫反应。

该图像是图表，展示了 WT、Tyk2-/- 和 Tyk2K923E 小鼠在 Candida albicans 感染后的中性粒细胞和单核细胞的浸润情况。图中表现为 IHC 染色的代表性图片以及流式细胞术分析，显示感染后不同基因型的小鼠中性粒细胞的比例变化，数据来源于独立实验，时间点包括感染后的第1、2和4天。

---

7. Conclusion & Reflections

7.1. Conclusion Summary

This study provides a comprehensive and rigorous investigation into the role of TYK2 in cutaneous fungal infections, leading to a novel and significant conclusion: TYK2 signaling is detrimental to the host during Candida albicans skin infection. This finding is contrary to the well-established protective role of TYK2 in many other infectious contexts.

The authors meticulously uncovered the underlying mechanism:

1. TYK2's kinase activity is essential for the production of the cytokine $IFNγ$ by skin-resident $γδ$ T cells in response to the fungal infection.
2. This localized $IFNγ$ signal acts on neutrophils, preventing them from forming a protective, barrier-like layer of necrotic cells around the fungus. Previous literature suggests $IFNγ$ can prolong neutrophil lifespan, which would explain the lack of necrosis.
3. Without this necrotic barrier, the fungus is better able to invade deeper skin tissues and disseminate systemically to distant organs like the kidneys.
4. Consequently, inhibiting TYK2's kinase function enhances host resistance by reducing $IFNγ$ levels, allowing the necrotic neutrophil barrier to form, thereby containing the infection locally and promoting faster healing.

7.2. Limitations & Future Work

The authors responsibly acknowledge several limitations of their study, which also point toward future research directions:

• Translation to Humans: The study was conducted exclusively in mice. While mouse models are powerful tools, the human immune system has differences, and it remains to be seen how directly these findings translate to human patients, especially those taking TYK2 inhibitors for autoimmune diseases.
• Specific Infection Model: The intradermal infection model mimics a deep skin wound, which is a specific clinical scenario. The role of TYK2 might differ in other types of candidiasis, such as superficial mucosal infections (e.g., thrush) or primary systemic candidiasis where the fungus enters the bloodstream directly. Future work should investigate TYK2's role in these other infection models.
• Direct In Vivo Proof for $γδ$ T Cells: While the evidence strongly points to $γδ$ T cells as the $IFNγ$ source, the final piece of evidence (in vitro stimulation) is not a direct in vivo confirmation. A future experiment using mice with a specific deletion of Tyk2 only in $γδ$ T cells would provide definitive proof.
• Mechanism of Necrotic Barrier Formation: The study visually identifies the necrotic barrier and demonstrates its functional importance through depletion experiments. However, the precise molecular mechanisms by which $IFNγ$ signaling prevents neutrophil necrosis, and how the necrotic layer physically impedes fungal invasion, are not fully elucidated and represent an area for further investigation.

7.3. Personal Insights & Critique

This is an excellent example of hypothesis-driven research that successfully challenges existing paradigms. The paper's greatest strength lies in its logical and systematic use of complementary genetic mouse models. The authors build their argument step-by-step, with each experiment logically leading to the next, creating a cohesive and convincing narrative.

• Clinical Relevance: The findings have immediate clinical implications. With TYK2 inhibitors becoming more common for treating diseases like psoriasis, this study suggests that such treatments are unlikely to increase the risk of cutaneous C. albicans infections and might even be beneficial. This is a crucial piece of information for the safety profile of these drugs.
• Context-Dependency of Immune Signaling: This work beautifully illustrates a fundamental principle of immunology: the function of a single molecule or pathway is highly context-dependent. TYK2 is not simply "pro-inflammatory" or "anti-pathogen"; its role is dictated by the specific pathogen, the tissue environment, and the cell types involved.
• Potential for Improvement/Critique:

  • The term "necrotic barrier" is compelling, but the paper could have strengthened the evidence for its role as a mechanical block. For instance, advanced imaging techniques could potentially visualize the interaction between fungal hyphae and this layer more dynamically.

  • The link between the RNA-seq data and the necrotic phenotype could be explored further. While the downregulation of ISGs is clear, the authors did not pinpoint a specific ISG or pathway that directly controls neutrophil death in this context. This remains an open question.

    Overall, this paper makes a significant contribution to our understanding of fungal immunity and the complex roles of JAK-STAT signaling. It not only solves a specific question about TYK2's function but also opens up new avenues of research into neutrophil biology and the therapeutic potential of modulating specific immune pathways during infection.