Introduction
Clinical manifestations of trichodermasis include pneumonia (1), rhino-orbital-cerebral mycosis, oral infection, otitis externa, sinusitis, brain abscess, stomatitis, mediastinitis and peritonitis (2), endocarditis (3), skin and skin structural infection, keratitis, and septic shock (4). Trichoderma spp. are often isolated from tumors. Much has been learned about the relationship between fungi and cancer. Among them, Aspergillus flavus is common, and its metabolite aflatoxin may lead to liver cancer. Trichoderma is similar to Aspergillus in morphology and evolution. There have been no published reports that Trichoderma or its toxin can induce carcinoma. We recently reported the isolation of Trichoderma longibrachiatum from the tissue of a patient with pulmonary spindle cell carcinoma that was confirmed as primary infection by laser capture microdissection and polymerase chain reaction (5). However, its potential role is unclear. To explore whether the strain can cause atypical cell proliferation, we designed a preliminary mouse infection experiment.
Materials and Methods
Isolates
The green filamentous fungus PKUT180420015 was isolated from the tissue of a patient with pulmonary spindle cell carcinoma at Peking University Third Hospital. The isolate was grown on Sabouraud's dextrose agar (SDA) at 28°C for 7 days. Conidia were harvested by gently washing the surface of the slants with saline. The suspension was filtered through a sterile 40 μm cell strainer, and the number of conidia was adjusted to 5 × 107 colony-forming units per milliliter (CFU/ml) by counting the spores in a hemocytometer and subsequently verifying these results through quantitative colony counts on PDA plates. Resting conidia were immediately used or stored at +4°C (6).
Mice
Twelve ICR mice (male, 18–22 g, 6–8 weeks) were purchased from Beijing Keyu Animal Breeding Center Company (Beijing). All mice were bred in the Animal Experimental Department, Fourth Medical Center, PLA General Hospital under specific-pathogen-free (SPF) conditions. The 12 mice were randomly divided into four groups, with three mice in each group.
Establishment of an Immunosuppression Model
Mice in Group 1, Group 2, and Group 3 were immunosuppressed by intraperitoneal injection of cyclophosphamide (80, 90, 100 mg/kg·d, respectively) for 3 days. Mice in Group 4, the control group, received an intraperitoneal injection of cyclophosphamide at 100 mg/kg·d (6).
Establishment of the Skin Infection Model
Mice in Groups 1, 2, and 3 between 6 and 8 weeks of age were infected by intracutaneous injection on the skin of the back with a suspension of T. longibrachiatum (0.02 ml). Group 4 was intracutaneously injected with saline (0.02 ml) as the normal control. To evaluate the effect of this study on the mental state of mice, we observed their appetite, water consumption and cage activity.
Access and Processing of Skin Specimens
At 3, 7, and 14 days after inoculation, one mouse was euthanized in each group by the atlantoaxial dislocation method. Skin specimens from the inoculation site were obtained for fungal culture in SDA at 28°C for 7 days The skin specimens of the mice 14 days after inoculation in Group 3, whose ulcer was the most typical, were embedded in paraffin and serially cut for histopathological examination (hematoxylin-eosin staining, periodic acid-Schiff staining, and periodic acid-silver methenamine staining).
Antibodies
The primary antibodies used for immunohistochemical staining included the following: anti-CD56/NCAM (neural cell adhesion molecule), anti-Ki67 and anti-granzyme B (all from Abcam, Boston, USA), anti-perforin and anti-CD3ε (all from Cell Signaling Technology, Danvers, USA). Anti-rabbit (Zhongshan Gold Bridge, Beijing, China) was used as the secondary antibody for immunohistochemical staining. Unstained cells were used as negative controls, and mouse spleen cells were used as positive controls.
Immunohistochemical Staining
The paraffin sections were dried at 60°C for 4 h, and paraffin was removed by immersing them into dimethylbenzene substitutes (3 × 15 min), absolute ethanol (2 × 5 min), 95% ethanol (2 × 5 min), and 80% ethanol (5 min) and then distilled water for 2 min. Then, the samples were incubated with 3% peroxide-methanol in a light-resistant container at room temperature for 10 min for endogenous peroxidase ablation. All following steps were carried out in a moist chamber: The samples were rinsed 3 times with clean water, 3 times with distilled water and 3 times in PBS (phosphate buffer solution, pH 7.4). Antigens were retrieved by high pressure and high temperature antigen retrieval via immersion in citric acid buffer (pH 6.0). The slides were rinsed in PBS for 3 × 5 min. The anti-CD56/NCAM, anti-Ki67, anti-CD3ε, anti-perforin and anti-granzyme B antibodies were diluted to 1:2,000, 1:600, 1:150, 1:600, and 1:1,200, respectively. They were dropped (50–100 μl) onto slides and incubated at 37°C for 2 h. Then, 50–100 μl of secondary antibody solution was dropped onto the slides and incubated for 30 min at room temperature. The slides were rinsed in PBS for another 3 × 5 min. All slides were incubated with 3,3-diaminobenzidine (DAB, Zhongshan Gold Bridge, Beijing, China) for coloration. The cell nuclei were counterstained with hematoxylin. Then, the slides were immersed in 95% ethanol (2 × 2 min), absolute ethanol (2 × 2 min), and dimethylbenzene substitutes (2 × 5 min). Finally, the slides were dehydrated and integrated with neutral balsam.
Discussion
Trichoderma Pers. is a fungus that exists in nature worldwide. Some species of Trichoderma spp. can inhibit pathogenic fungi; thus, it is widely used in agricultural production as a biological control agent (7). Since the first case of Trichoderma infection was reported in 1970 (8), trichodermasis has now included pulmonary infection, rhino-orbital-cerebral mycosis, oral infection, otitis externa, sinusitis, brain abscess, stomatitis, peritonitis, endocarditis, skin and skin structural infection, keratitis, and septic shock. However, Trichoderma infection-associated cancer has not been encountered. We recently reported the isolation of Trichoderma longibrachiatum (PKUT180420015) from the tissue of a patient with pulmonary spindle cell carcinoma that was confirmed as a primary infection by laser capture microdissection and polymerase chain reaction (5).
According to the European Organization for Research and Treatment of Cancer and the National Institute of Allergy and Infectious Diseases consensus definitions, our case fulfills the criteria for a “Proven” invasive fungal infection (deep fungal infection) by T. longibrachiatum: histopathologic examination showing hyphae from a biopsy specimen with microscopic evidence of associated tissue damage; and growth of T. longibrachiatum from a sample obtained by a sterile procedure from a normally sterile and clinically or radiologically abnormal site consistent with infection (microbiological criteria). Periodic acid-Schiff staining, periodic acid-silver methenamine staining, and Calcofluor staining showed fungal spores in the vascular lumen, vascular walls and around the blood vessels. In addition, deep fungal infection caused by T. longibrachiatum was later confirmed by laser capture microdissection and polymerase chain reaction (5).
Immunohistochemistry showed positive markers of Ki67, CD3, CD56, GZMB, and PRF, which suggested NK-cell or T-cell infiltration. CD3 is mainly involved in the signal transduction of T cells and consists of four polypeptides, ζ, γ, ε, and δ; thus, CD3ε is mainly used to label T lymphocytes (9). CD56 plays an important role in embryonic development and the interconnection of nerve cells and is used to identify NK cells, but some cells with T-cell markers CD3 and CD4 also express CD56 (10). Perforin is a pore-forming protein that promotes the entry of granulase and other cytotoxic serine proteases into target cells. Perforin is mainly expressed in cytotoxic T lymphocytes and NK cells (11). Granzyme B belongs to the family of serine proteases expressed by cytotoxic T lymphocytes and NK cells and is a key component of immune responses to pathogens and transformed cells (12). Ki67 can be used to detect proliferating cells in G1 phase, S phase, G2 phase and mitotic phase but not in G0 resting phase and is mainly used for evaluating tumor cell proliferation (13). Therefore, Trichoderma infection leads to the aggregation of T cells and NK cells, which are involved in the inflammatory process of Trichoderma infection.
NK/T cells are important for host resistance to microbial infection. Recent studies have shown that these cells can aggregate or proliferate in Mucor irregularis and Rhizopus arrhizus infections that resolve after antifungal therapy alone (14, 15). Further studies showed that Mucor irregularis and Rhizopus arrhizus could induce NK/T-cell infiltration (CD3+, CD8+, CD56+, TIA1+, GZMB+, PRF+), proliferation (Ki67+), and angioinvasion in mice (6, 14). Recently, Schmidt et al. demonstrated that both interleukin-2-prestimulated and unstimulated human NK cells could damage Rhizopus oryzae hyphae, which corresponds with mucoramycosis (16, 17). The case presented here showed that the Trichoderma longibrachiatum strain (PKUT180420015) could also induce NK/T-cell infiltration.
With the increasing number of immunodeficient patients in recent years, the spectrum of opportunistic pathogenic fungi has expanded, and Trichoderma spp., which were once considered non-pathogenic, are becoming increasingly worthy of clinical attention. Trichoderma infections usually occur in immunocompromised patients. Among immunodeficient patients, Trichoderma strains can cause various diseases ranging from local infections to fatal disseminated diseases. Our study shows that PKUT180420015 isolated from lung tumors can induce mice to express CD3, CD56, perforin, Granzyme B and Ki67 markers. Further investigation of the mechanism of Trichoderma longarm-related proliferation is needed.
Trichoderma spp. are resistant to amphotericin B, itraconazole, ketoconazole and other traditional antifungal drugs (18). Therefore, it is necessary to actively carry out drug sensitivity tests for Trichoderma infection in clinical practice.
In summary, our studies have shown that the Trichoderma longibrachiatum strain (PKUT180420015) isolated from a biopsy specimen of a patient with pulmonary spindle cell carcinoma could induce atypical hyperplasia, with the expression of Ki67, CD3, CD56, GZMB, and PRF in mice, indicating that the fungus may be involved in inducing tumorigenesis.