Effect of CAP10 gene on immune response in mice infected with Cryptococcus neoformans

ABSTRACT Background Capsule is an vital virulence factor in Cryptococcus neoformans infection. Recent studies show CAP10 is a key gene in capsular formation. However, the role of CAP10 in the pathophysiology of cryptococcosis is still not well understood. This study aims to investigate the association of CAP10 expression with the immune responses to infected mice. Methods The shRNA expression plasmid was designed to interfere with the synthesis of CAP10. The animal model was established with C. neoformans wt strain H99, cap10-shRNA C. neoformans and PBS control in the respiratory tract. On the 7 days and 21 days after infection, mice lung histopathological examination and homogenate culture were performed, and cytokines expression level in the serum of mice were quantitatively detected. Results The lower degree of edema and infiltration of inflammatory cells were observed in cap10-shRNA group. The growth rate of cap10-shRNA strain was significantly reduced. In addition, interference with CAP10 altered the expression profile of Th1, Th2, Th17 and Treg type cytokine. Down-regulation of CAP10 was beneficial to the balance of Th1/Th2, Th17/Treg ratio. Conclusions Taken together, our results indicated the expression of the CAP10 was associated with an antifungal immune response to mice infected with C. neoformans. CAP10 might play an important role in regulating the inflammatory response, and could expected to be a new molecular therapeutic target in cryptococcosis. CFU: Colony-forming units; ELISA: Enzyme-linked immunosorbent assay; IFN-γ: Interferongamma; IL–4: Interleukin–4; IL–2: Interleukin–2; IL–17: Interleukin–17; TGF-β: transforming growth factor-β; CAP10: The capsule associated protein 10 gene.

and Cytokines produced by T cells play an important role [1][2][3] .The development and outcome of cryptococcosis is not only affected by the immune status of the host, but also depends on the expression differences in various virulence factors, such as polysaccharide capsule, phosphatidylserine synthesis 4 , laccase, urease and melanin.Among them, the high molecular weight polysaccharide capsule is the most Classical virulence factors, contributing approximately 25% of the total virulence composite [5][6][7] .However, the relationship between capsular virulence factors and its role in the pathophysiology of cryptococcosis is still unclear.
The capsule associated protein 10 gene (CAP10) is a key gene in capsular formation.There is a correlation between capsular thickness and CAP10 expression 8 .Studies have shown that knocking out the CAP10 in wild-type Cryptococcus can cause capsular loss, which shows no pathogenicity in animal models.If recomplemented with the CAP10, the strain forms a capsule and shows toxic in animal models 9 .Some metal ions, such as Mg2+ that have an effect on CAP gene expression, which are also important to capsule biosynthesis and virulence 10 .A mutation in the CAP10 results in a pleiotropic phenotype with cell size, expression of virulence factors and size of extracellular vesicles 11 .Our previous studies showed that the RNA interference on the expression of CAP10 gene can induce phagocytosis of C. neoformans.In this study, an animal model was established with inoculating C. neoformans wt strain H99 or cap10-shRNA C. neoformans through respiratory tract.Our objective was to determine whether CAP10 expression affects the course of pulmonary infection and dissemination in infected animal.Understanding these functions of CAP10 in host immune response may provide opportunities to improve the effective control and treatment of cryptococcosis.

Plasmid and strain
Psilencer4.1-CMV neo was purchased from Invitrogen Inc. C. neoformans strain (wt strain H99) was purchased from cryptococci professional laboratory of China Medical fungus preservation management center, E. coli DH5α was purchased from Tiagen Inc.

Construction, connection and transformation of shRNA expression vectors
According to the CAP10 nucleotide sequence provided by the GenBank database, the targeting sequence was designed using Ambion's siRNA online design software.To clone the siRNA into the vector psilencer4.1-CMVneo and express the shRNA by this vector, the sticky end of HindIII and BamHI were used to form the expression cassette.The full sequences used were as follows: sense 5′-GATCCGGGCTTTGATGAATGGTACTTCAAGAGAGTACCATTCATCAAAGCCCTTA-3′ and antisense 5′-AGCTTAAGGGCTTTGATGAATGGTACTCTCTTGAAGTACCATTCATCAAAGCCCG-3′.Two microliters of each oligo was combined in a 50 μL reaction system.The oligos were denatured at 95°C.The temperature was decreased by 5°C every 5 minutes until 45 °C, so that the single strand is anneal to form a double strand.Plasmid psilencer4.1 -CMVneo was digested by endonuclease HindIII and BamHI, and then Annealed oligos were ligated into the HindⅢ and BamHI sites of psilencer4.1-CMVneo.The recombinant plasmid was transformed to the competent E.coli DH5α, then the positive clones were selected, and verified by sequencing.

Transduction and selection of stable clones
The recombinant plasmid was transfect to C. neoformans using the LiAc lithium acetate yeast transformation kit.Stable C. neoformans expressing cap10-shRNA was selected using 100 μg/ml G418 in YPD.The culture medium was changed every 2~3 day, and transfect C. neoformans was harvested with G418 after 14 days of selection.

Experimental animal
Inbred strain mice are the most sensitive animals to Cryptococcus neoformans.SPF graded female C57BL/6 mice were purchased from the Lecco Experimental Animal Center of Fujian Province.96 mice were randomly divided into 3 groups, with 32 mice in each group.Mice were 8~12 weeks of age and the body weight was 16~22g at the time of infection.Rat group breeding cage was used and SPF grade mouse feed provided by Beijing Huayueyang Biotechnology Co., Ltd.All studies were conducted according to a protocol approved by the experimental animal ethics committee of fujian medical university.All studies were in line with the requirements of the "National Laboratory Animal Management Regulations" for the care and use of animals.

Preparation of fungal suspension
C. neoformans wt strain H99 group and the cap10-shRNA strain group were washed with PBS, then were centrifuged at 1200r /min for 5min and resuspended in sterile double distilled water.The number of C. neoformans were counted on the counting plate and adjusted to a final concentration of 5×10 6 CFU/mL.

Mouse model of cryptococcus neoformans infection
Sodium pentobarbital is formulated to a concentration of 3% before use.Injection dose is 30mg/kg.Then observe the anesthetic effect until the eyelash reflex disappears and the muscles relax, reaching the anesthesia requirements of this experiment.To simulate the natural infection process, the mice were quickly inoculated with 20μL of the fungal suspension through unilateral nostrils after anesthetized with Sodium pentobarbital intraperitoneal injection.Three experimental groups were inoculated separately with C. neoformans wt strain H99, cap10-shRNA C. neoformans or equal amounts of PBS solution as control.

Mouse lung histopathological examination and homogenate culture
Mice were euthanized with on 7 th and 21 th day post-inoculation in each group.We took the utmost care to alleviate suffering of the mice.Intraperitoneal injection of sodium pentobarbital, the dose is generally about 3 times the anesthetic dose.Judging the death of experimental animals: continued without spontaneous breathing for 2-3min and no blink reflex.Then lung tissues were excised using an aseptic technique.Lung tissues were fixed and immersed with neutral buffered formalin for 24h, and then placed into TS12C automatic dehydrator, embedded in paraffin at 60 °C.5μm-thick slices of organs were cut and stained with PAS method.Slices were analyzed using light microscopy.In a separate experiment,lung tissues were homogenized in sterile PBS, followed by cultured on YPD agar.CFU were counted following incubation at 30°C.

ELISA
The ELISA double-antibody sandwich method was used for the detection.The Mouse Quantikine ELISA Kit R&D Systems, America was purchased from Xiamen Taijing Biotechnology Inc.The test procedure is strictly followed according to the reagent instructions.Serum samples of three groups of mice were obtained in the same manner, and the levels of Treg/Th17 and Th1/Th2 related cytokines were determined by ELISA, including interleukin-2 (IL-2), gamma interferon-γ (IFN-γ), interleukin-4 (IL-4), transforming growth factor beta (TGF-β), and interleukin-17 (IL-17).

Statistical analysis
All data were expressed as mean ± SEM for each experimental group.Statistical significance was calculated using Student's t test of individual paired comparisons.A P value < 0.05 was considered to be statistically significant.Analyses were performed using GraphPad Prism version 6.00 for Windows (GraphPad Software, San Diego California USA).

Construction and verification of stably expressing cap10-shRNA
Following transfection with cap10-shRNA plasmid, C. neoformans cells were selected using G418 at 100 μg/ml.After 2 weeks, non-transfected cells were completely dead, and G418 resistant colonies were harvested and grown in culture medium containing 50 μg/ml G418.
The recombinant plasmid was sequenced by an automatic gene sequencer (Invitrogen company), and the results were consistent with the design sequence.FQ-PCR was performed to verify that CAP10 gene expression in stably transfected cells was suppressed by cap10-shRNA.The experimental details were described previously 12 .

The cap10-shRNA strain alleviated pathological lesions in pulmonary C. neoformans infection
In order to examine the levels of pulmonary inflammation in these three groups of animals, we picked the examination time points of day 7, and day 21 post-infection (The average survival time of mice with cryptococcosis was about 28 days, so we chose day 7 as early infection points and day 21 as later infection points).
The paraffin-embedded specimens and the pathological section of the lungs were observed.On day 7, compared with the control group, both the wt group and the cap10-shRNA group had swollen lung tissues.The wt group of C. neoformans showed obvious enlarged edema in the lungs.The degree of edema in the cap10-shRNA group was lower than that in the wt group (Fig. 1A).Pathological sections of lung tissue showed that on the 7th day after infection, a large number of inflammatory cells infiltrated in the wt strain, and numbers of C. neoformans distributed among the whole lung.Some granuloma and tissue necrosis began to form.In the cap10-shRNA group, scattered Cryptococcus was observed, and the degree of infiltration of inflammatory cells was lower than wt group.No obvious granuloma and necrosis was observed (Fig. 1B).On day 21, the degree of edema in the wt group was further increased, while the change in the cap10-shRNA group was not significant compared with the time points of day 7 (Fig. 1A).In the wt strain, the granuloma gradually enlarged to invade the surrounding tissues, and the fibrous tissue proliferated.Later, the lesions may be surrounded by fibrous tissue or fibrous scars.There were a large number of macrophages and lymphocytes inside.In the cap10-shRNA strain, only a few lymphocytes and neutrophils were infiltrated, while the necrosis and granuloma were still not obvious (Fig. 1B).

Inhibition of cap10 expression reduced lung fungal burden in infected mice
Pulmonary growth and clearance of wt strain were compared to those cap10-shRNA strain.
Mice were inoculated intranasally with a final concentration of 5×10 6 CFU/mL, and the pulmonary fungal burden kinetics was evaluated.The wt strain showed continuous growth in lungs, resulting in the burden of C. neoformans expansion during 3weeks [CFU(log10):week 1, 5.52; week 3, 6.20].This was completely different contrast with the cap10-shRNA strain, which showed a slight decrease during 3 weeks (CFU(log10):week 1, 2.83; week 3, 2.68).Burden in the group infected with the cap10-shRNA strain was about 1000-fold lower than that of the group infected with the wt strain at the end of week 3(Fig.2).These results may suggest that the growth and clearance of cryptococcus in the lungs is related to cap10

Inhibition of cap10 expression altered cell-mediated immune response in infected mice
IFN-γ and IL-2 are Th1-type cytokines, IL-4 is a Th2-type cytokine, IL-17 is a Th17-type cytokine and TGF-βis a specific cytokine produced by Treg cells.So we further observed these cytokine profiles in infected mice serum on day 7, and 21 post-infection.Compared with the PBS-control group, the Th1 type cytokine IFN -γ and interleukin-2 (IL-2) levels in mice infected with the cap10-shRNAstrain and in those infected with the wt strain were significantly higher at early phase.The Th1-type cytokine IFN-γ and interleukin-2 (IL-2) levels in the cap10-shRNAstrain still maintained a relatively high level in later phase.
Compared with wt strain, the Th1-type cytokine levels showed significantly higher and Th2-type cytokine showed significantly lower in later phase in the cap10-shRNAstrain(Fig.3A3B3C).It implied that inhibition of cap10 expression might induce protective cell-mediated immunity in infected mice.The Th2-type cytokine interleukin-4 in the cap10-shRNAstrain and in wt strain was significantly higher than control in early phase and later phase.It is suggested that interference with CAP10 can alter the expression of Th1 and Th2 type cytokine.As for interleukin-17, it showed significantly higher in the cap10-shRNAstrain and in wt strain compared with control group at each time point.As for transforming growth factor beta (TGF-β), it was significantly lower in the cap10-shRNAstrain and in wt strain compared with control in early phase, but significantly higher in the cap10-shRNAstrain compared with control in later phase(Fig.3D3E).It implied that in the later stage of infection, the higher concentration of TGF-β maybe induced the conversion of CD4+T cell into Tregs, which can alleviate the immune damage caused by Cryptococcus, thereby achieving the purpose of protecting the host.

Inhibition of cap10 expression altered the balance of Th1-Th2 and Th17-Treg responses
The immune response pattern is reflected by the ratio of Th1 to Th2 cytokines (Th1/Th2) and Th17 to Treg cytokines (Th17/Treg).Compared with the PBS-control group, the IFNγ/IL-4 ratio and the IL-2/IL-4 ratio showed significantly lower in the cap10-shRNAstrain and in wt strain at later time point.Compared with the cap10-shRNA strain, the IFN-γ/IL-4 ratio and the IL-2/IL-4 ratio showed significantly lower in the wt strain at later time point(Fig.4A4B).It indicated that Th2-based immunity should be the main way for C. neoformans to escape host clearance.As the disease progressed, the Th1/Th2 immunity imbalance became more and more serious in wt strain.As for the ratio of IL-17/TGF-β, compared with the PBS-control group, it was significantly higher in the cap10-shRNAstrain and in wt strain at early time point, but only wt strain still showed significantly higher at later time point.Compared with the cap10-shRNA strain, the IL-17/ TGF-β ratio showed significantly higher in the wt strain at each time point (Fig. 4C).It implied that the Th17 response is dominant in the early phase, but with the development of disease, the secreted TGF-β cytokine maintained high levels in cap10-shRNA strain.

Discussion
The capsule of C. neoformans regulates the host's immune response from many levels, which including changing the antigen presentation process to make the antibody nonresponsive, cytokine secretion disorder, anti-phagocytosis, capable of absorbing microbicidal oxidative bursts, white cell migration inhibition 13,14 .In this study, the effect of down-regulation of CAP10 expression of the pathophysiological process of C. neoformans infection was observed.The cap10-shRNA strain was found smaller distributed among lung tissues, less inflammatory cell infiltration and no obvious necrotic lesions to be observed on 7 days (FIG.1).The wt strain was showed a sustained strong inflammatory response with the gradually enlarged granuloma and the fibrous tissue proliferated, even invaded into the surrounding tissues on 21 days.It demonstrates that the cap10-shRNA strain may alleviate pathological lesions in pulmonary C. neoformans infection.The cap10-shRNA strain was also found to exhibit a retained ability to grow on the YPD agar, though the growth rate was significantly reduced (FIG.2).In contrast, a progressive growth of the wt strain was found on the YPD agar.
Cytokine patterns of the serum may be related to clinical outcomes [15][16][17] .Gressler and colleagues reported an association between Th1/Th2 cells and fungal growth and dissemination in Cryptococcosis caused by Cryptococcus neoformans 18 .Th1-typed cytokines may induce prolonged STAT1 binding and protection against C. neoformans 19 .
Additionally, Darin also reported that regulatory T cell induction and retention in the lungs drives suppression of detrimental Th2 cells during pulmonary cryptococcal infection 20 .We therefore focused on the cytokine profiles after inoculated with pathogenic Cryptococcus strains.From the results of the cytokine profiles (FIG.3), we found the production of Th1 cytokines were reduced while Th2 cytokines were increased at later time point in wt strain, but Th1 and Th2 cytokines in cap10-shRNA strain both increased.Microbial infections such as bacteria, fungus and viruses may also cause Th1/Th2 imbalance in vivo.
A significant drift of the Th1/Th2 cytokines ratio at later time point was observed in the WT group (FIG.4).It suggested that the Th2-type immune response might be the main way for C. neoformans to escape host clearance, which might make cryptococcal cells unable to be effectively cleared by phagocytic cells.The fungus could further spread or even infect the central nervous system by means of the "trojan mechanism".Th17 cells also play an important role in infectious disease through their secreted molecules.Th17 cells and its secreted cytokines IL-17 are responsible for the immune surveillance, but also involved in the pathogenesis of many autoimmune diseases and in the mechanisms of fibrosis in many organs after the infection [21][22][23] .Unlike Th17 cells, Treg cells are a subset of T cells with immunosuppressive effects, which control the intensity of immune responses and reduce the damage of inflammatory responses to tissues 24 .The imbalance between Th17 cells and Treg cells is an important factor in the pathogenesis of many inflammatory and autoimmune diseases [25][26][27] .From our results, we found IL-17 maintained a relatively high level in wt and cap10-shRNA strain at each time point while TGF-β showed significantly higher in the cap10-shRNAstrain compared with control at later time point.As for the ratio of IL-17/ TGF-β, compared with the cap10-shRNA strain, the IL-17/ TGF-β ratio showed significantly higher in the wt strain at each time point.Compared with PBS control, only wt strain showed significantly higher at later time point.
TGF-β plays a key role in the balance between Treg cells and Th17 cells.It has been reported that low concentration of TGF-β can induce the expression of ROR-γt, but high concentration of TGF-β can inhibit the expression and function of ROR-γt and facilitate the production of Treg cells 28 .So that the increased Treg cells could regulate the balance between the response of effector T cells to remove pathogens and their over-immune response to severe inflammation and tissue damage.This observation matched well with pathological examination, less granuloma and necrosis were observed in the cap10-shRNA strain on 21days.Conclusions This study clarifies the relationship between the immune response pattern and the CAP10 gene in mice infected with C. neoformans.The amount of CAP10 expression can affect the secretion of cytokines.Knocking down the cap10 gene contributes to the balance of Th1/Th2 and Th17/ Treg immunity during the development of cryptococcosis.It is also beneficial to controling the spread of C. neoformans, which can affect the prognosis of cryptococcosis.A better understanding of the relationship between the immune response pattern and the CAP10 gene is helpful in findinga novel molecular therapeutic target to improve the outcomes of patients with cryptococcosis.

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