Dermatophytosis is an unsolved worldwide public health problem. In immunocompetent people, it usually results in superficial infections and is not fatal. However, it is easy to relapse and reinfection, resulting in a huge economic burden and harm to patients’ quality of life. In 2017, the United States spent more than $800 million on the treatment of human dermatophytosis (1).

The inflammatory response to a certain dermatophyte infection varies from host to host. Humans are generally susceptible to anthropophilic dermatophytes, such as Trichophyton rubrum. The infection always accompanies by mild inflammation and tends to be chronic, easy to relapse, and unable to heal itself (2). While mice, the most commonly used experimental animals, are resistant to many species of dermatophytes, including Trichophyton rubrum, Trichophyton mentagrophytes, and Microsporum canis. Even if the experimental infectious model is constructed under certain conditions, the infection always results in severe inflammation and the pathogens were eliminated about a month after inoculation (3–5).

It has long been recognized that inflammation to dermatophyte infection is different among various hosts. While the mechanism underlying is still not well understood. A recent study has shown that even mice with combined T and B lymphocyte defects can eliminate the dermatophytes spontaneously (3). This suggested that innate immunity plays an important role in the host’s inflammatory response and outcome to dermatophyte infection.

Toll-like receptor 2 (TLR2), a single transmembrane pattern recognition receptor, consists of an extracellular domain at the N terminal, a transmembrane domain, and a conserved TIR domain at the C terminal. The extracellular domain mediates the recognition of specific pathogen-associated molecular patterns (PAMPs). After recognition of its ligand, lipopeptide, TLR2 activates mitogen-activated protein kinase (MAPK) comprising p38, JNK, ERK, and the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway through Myd88-dependent pathway (6, 7), followed by regulating the expression of cytokines and chemokines (8). It plays an important role in phagocytosis and pro-inflammatory responses against dermatophyte infection (9, 10).

Although TLR2 is a conserved protein, there are still some differences in its sequences and spatial conformations between humans and mice, especially in its extracellular ligand recognition region (11). Therefore, we speculate that the interspecific difference of TLR2 ectodomain resulted in the different inflammatory manifestations between humans and mice infected by dermatophytes. To verify this hypothesis, we replaced the extracellular domain of human TLR2 with that of the mouse by infusion clone to construct a mouse-human fusion TLR2 expression vector, and then re-expressed it into a TLR2 knockout HaCaT strain to construct mice-human fusion TLR2 expressed HaCaT (mhTLR2-HaCaT). Meanwhile, the human TLR2 vector was re-expressed into the TLR2 knockout HaCaT to construct human TLR2 re-expressed HaCaT (hTLR2-HaCaT). Trichophyton rubrum and Microsporum canis were chosen as the subjects of our study since Trichophyton rubrum is a representative anthropophilic dermatophyte and is the most predominant causative agent of human dermatophytosis. And Microsporum canis is a representative zoophilic dermatophyte and is the most predominant causative agent of dermatophytosis in domesticated pets such as dogs and cats. Then hTLR2-HaCaT and mhTLR2-HaCaT were co-cultured with Trichophyton rubrum and Microsporum canis respectively to explore the interspecies differences of TLR2 in recognition and inflammatory response to these two dermatophytes.

Generally, the dermatophytes were confined in the epidermis. As the constructed cells of the epidermis, keratinocytes can recognize and perceive PAMPs of invasive dermatophytes through pattern recognition receptors on their surface, such as TLR2, and trigger the inflammatory responses by activating downstream signaling pathways (10, 12, 16, 17). HaCaT cell, the immortalized keratinocytes cell line, is the well-established research model of human keratinocytes to simulate dermatophyte infection (10, 16, 18, 19). Therefore, HaCaT was used to carry out our study.

Innate immune response to fungi is mainly mediated by MAPK and NF-κB signaling pathways (20–22). While, different pathogenic fungi or even the different morphological forms of fungi will activate their preferential pathways to a certain degree (19, 21). As for dermatophyte infection, it seems that the MAPK pathway, rather than the NF-κB pathway, plays a more important role in antifungal immunity (14). By the global gene expression analysis, we found that c-Jun was the core gene when the HaCaT was challenged with M. gypseum. Further immunoblotting showed that Jun N-terminal protein kinase (JNK) and p38 pathways were significantly activated. While these pathways were largely unchanged when the HaCaT was challenged with the anthropophilic T. rubrum (19). Achterma et al. (12), combined with primary keratinocytes and organotypic epidermal models also found that two branches of the MAPK signaling pathway, p38, and JNK, were activated by Trichophyton equinum. Afterward, the cascade activation of the MAPK pathway leads to the secretion of IL-1α, GM-CSF, and IL-8. While, the NF-κB pathway was largely unchanged and the other major MAPK pathway, extracellular signal-regulated kinases 1 and 2 (ERK1/2), was even inhabited.

In the present study, we found that after being infected by Trichophyton rubrum, the p38 signal pathway was activated in mhTLR2-HaCaT cells, and the JNK/c-Jun signal pathway was significantly activated after Microsporum canis infection, while these two pathways were unactivated when hTLR2-HaCaT cells were challenged by each of the two dermatophytes. This indicated that p38 and JNK pathways in human keratinocytes against these two dermatophytes are active after replacing the extracellular domain of human TLR2 with that of mouse TLR2. The most reasonable explanation for this phenomenon is that the spatial structure of the extracellular ligand recognition region of mouse TLR2 is more prone to recognize the PAMPs of these two dermatophytes to activate the downstream signal pathways.

Jin et al. (11), found that although the backbone structures of the human and mouse TLR2s are practically identical, their side chains in the lipid-binding pocket differ significantly in sequences and structures. The sequence differences, including Thr335 to Leucine, Pro306 to Leucine, and Leu266 to Phenylalanine cause the shape of the lipid-binding pocket to differ substantially between mouse and human TLR2s. This resulted in more efficient recognition and binding of lipopeptides with shorter lipid chains by mouse TLR2 than human TLR2. So, it implies that the PAMPs of these two dermatophytes may contain short lipid chain lipopeptides, which were prone to be recognized by the mouse TLR2, not the human TLR2.

By the way, our results also show that different dermatophytes have their preferred signaling pathways, the infection of Trichophyton rubrum is more likely to cause the activation of the p38 pathway, while the infection of Microsporum canis is more likely to activate the JNK/c-Jun pathway. This is likely to be one of the mechanisms by which the same host produces different inflammatory responses after infection with different dermatophytes.

The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

JC, conceptualization, funding acquisition, and writing –original draft; ZS, methodology and writing – review & editing; WD and SZ, resources and methodology; ML and SY; resources. All authors contributed to the article and approved the submitted version.