Introduction

Infection can come from different types of pathogens and microscopic organisms, such as bacteria, viruses, parasites and fungi. The article will focus on fungal infections. 

Fungal bacteria take advantage of a weakened immune system and infect the host. This can range from athlete’s foot and yeast infections to severe, life-threatening conditions such as aspergillosis. Therefore, the body must have strong innate immunity as the first line of defence against fungal infections. 

The innate immune system is composed of macrophages, neutrophils and natural killer cells. These components work together to identify and eliminate the invading fungal bacteria. They do so by using pattern recognition receptors (PRRs).¹

CARD9 is the key signalling protein in the innate immune system. Its primary function is to detect specific fungal infections and relay the signal from the PRRs to other receptors, which activate the immune response. The purpose of this article is to explore how deficiencies in CARD9 affect antifungal immunity.²

What is CARD9?

CARD9 is short for Caspase Recruitment Domain-containing protein 9. It is a signalling adaptor protein, responsible for recognising fungal infections and stimulating immune cells to initiate the inflammatory response. 

This protein is mainly expressed in myeloid cells, such as:

• Macrophages - a type of white blood cell which engulfs and digests pathogens
• Dendritic cells - specialised immune cells that present antigens to other immune cells
• Neutrophils - first responders to infection and inflammation 

CARD9 is also found in;

• Endothelial cells 
• Lungs
• Heart
• Brain 
• Bone marrow²

This protein is involved in several pathways within the immune response. It acts at various points downstream of the signalling pathway, which leads to the activation of the PRRs for the inflammatory response.²

CARD9 mechanism

• CARD9 acts as an intracellular adapter molecule, acting as a pillar which brings the proteins of the signalling pathway together. However, it has no enzymatic activity of its own
• Once CARD9 has activated PRRs, the NF-kB and MAPK signalling pathways are activated. These two mechanisms are central to inflammation and the immune response
• Another pathway activated by CARD9 is the production of inflammatory cytokines and chemokines. These molecules recruit immune cells by triggering cell migration, bringing immune cells to the site of infection 
• C-type lectin receptors (CLR), a type of PRR, form a complex with Dectin-1, which is then activated by CARD9 and stimulates inflammatory cytokine production

These pathways work in unison to combat infection. CARD9 is crucial in controlling the intensity of the immune response by mediating PRR signals.²

The role of CARD9 in antifungal immunity

Fungal bacteria are capable of hijacking a weakened immune system, thus infecting the body. Therefore, the immune system must be at optimal efficiency to keep fungal infections at bay. 

The receptors on immune cells can detect fungal cell wall components, which trigger CARD9 signalling. It recognises the fungal pathogens on PRRs, such as C-type lectin receptors.³

Following the activation of CLRs, the NF-kB pathway is activated by forming signalling complexes. This sets up the core transcription factors required for the inflammatory response, specifically, the production of pro-inflammatory cytokines such as:

• IL-1ß 
• IL-6
• IL-17
• TNF-alpha
• CXCL1

These components are required to attract immune cells to the area of infection, such as neutrophils.³

CARD9 ensures that the mucosal systemic immune responses work accordingly against fungal infections. These systems trap the fungal infection at the surface of endothelial cells, which line organs, and prevent its spread to deeper tissues.³

The mucosal immunity has 3 main aspects:

• Barrier defence activation: At mucosal surfaces, the moist inner lining of cavities and organs, CARD9 helps the immune cells in the area recognise any abnormal fungal elements through PRRs
• CARD9 is crucial for maintaining the strength of the mucosal barrier at endothelial cells. It initiates the production of IL-6 and IL-17, which stimulate the secretion of antibacterial proteins
• Neutrophils are the first line of defence for fungal invaders. CARD9 triggers neutrophil migration to the infected site by activating cytokine production³

CARD9 orchestrates these functions with systemic immunity, which involves:

• The activation and mobilisation of neutrophils in the bone marrow is supported and enhanced by CARD9 interactions
• During fungal infections, the body can build a wall made of granuloma cells. This can block off and contain invasive fungal infections. CARD9 helps to organise these cells
• CARD9 bridges the communication between detection by mucosal immunity and the required activation by systemic immunity. Furthermore, this alerts the rest of the body and initiates body preparedness³

CARD9 has an overarching role in several key pathways that ensure fungal immunity. 

Consequences of CARD9 deficiency

CARD9 deficiency is a genetic immune condition where a person becomes more vulnerable to fungal infections. The main fungi involved are:

• Candida 
• Aspergillus 
• Phialophora 

It is a disorder where the CARD9 gene is altered. This affects the production of the CARD9 protein. This creates abnormal CARD9 proteins in the neutrophil and macrophage cells. As a result, their ability to recognise foreign fungal infections is impaired, allowing for fungal diseases to develop. 

CARD9 deficiency is an inherited condition through an autosomal recessive inheritance. This is when the individual inherits two copies of the mutated gene, one from each parent.⁴

CARD9 deficiency has global consequences throughout the body:

• Fungal infections can range from superficial infections in the skin and nails, to more aggressive infections that affect organs, the brain and lymph nodes
• Neutrophil function is disrupted in CARD9 deficiency. They have a reduced ability to kill specific fungi
• Impaired neutrophils and macrophages result in reduced cytokine production when exposed to fungi
• The formation of granuloma cells is affected by the absence of CARD9⁴

Clinical manifestations of CARD9 deficiency

Individuals with both parents carrying the defective CARD9 gene have a 25% chance of their offspring showing clinical manifestations of CARD9 deficiency. Various fungi present specific clinical manifestations in CARD9 deficiency. 

• Chronic Mucocutaneous Candidiasis (CMC) is the recurring infection of the skin, nails, mouth, throat and genitals
• Invasive Candida is when Candida infections spread beyond superficial sites to more serious areas, such as the brain, eyes, bones, etc
• Deep Dermatophytosis is when fungal infections reach past the skin and infect deeper tissues, such as lymph nodes. 
• Other rare fungal pathogens, like Aspergillus, can infect the body in CARD9 deficiency

A key feature of CARD9 deficiency is the recurrence of fungal infections despite the use of antifungal treatments. This can become apparent anytime from childhood to adulthood.⁴

Diagnosis and management

People with recurrent fungal infections are flagged for possible CARD9 deficiency. The doctors will focus on the patient’s history of fungal infections and assess various factors:

• Type 
• Location 
• Severity 

Next, a series of laboratory tests is done on the patient’s blood sample. This is to assess the immune function of tissues and the microbiological components of the blood. To further confirm the diagnosis, genetic tests are carried out. This aims to identify the mutated CARD9 gene through targeted gene sequencing. 

After an agreed-upon diagnosis, the next step is to manage the condition. Several strategies can be taken:

• Antifungal therapy can be specifically adjusted to the type of fungal infection and the patient’s response
• Localised fungal masses can form in patients with CARD9 deficiency. Surgical intervention is required to remove the invasive infection
• Adjuvant therapies are used in specific cases where the infection is persistently returning. GM-CSF or G-CSF therapies are considered. These are colony-stimulating factors. This consists of cytokines, which are signalling molecules for white blood cell production. They specifically impact neutrophils but can have slightly varying targets. As a result, CARD9-driven activation can be assisted⁴

When deciding on the best management strategy, there are important considerations to be taken. The age of manifestation of CARD9 in the patient may require different handling. Furthermore, the specific clinical manifestations must be considered.⁴

Research and future directions

Researchers are currently exploring the mechanisms and potential therapeutic targets of CARD9 deficiency. Investigating this condition will prove to be tedious, as each type of fungal infection varies in mechanism and interactions. 

Using species-specific mechanisms to create targeted therapies for individual patients can be an area of interest. Additionally, immune cells and signalling pathways can be targeted and intercepted to fix any disruptions created by CARD9 deficiency.⁵ 

CARD9 immunity varies in different tissues, which impacts the clinical manifestations of the fungal infections in individuals. More research is being done in this area to understand the extent to which areas are affected. Clinical trials must be carried out to evaluate the potential of new therapies in different tissues with different species of fungi.⁵

Further genetic studies are needed to understand the diversity of CARD9 mutations and their specific consequences. The development of the disease must also be tracked to identify any therapeutic targets.⁵