Phagocytosis and nitric oxide production by peritoneal adherent cells in response to Candida albicans in aging: a collaboration to elucidate the pathogenesis of denture stomatitis

Abstract Elderly denture wearers are commonly affected by Candida-associated denture stomatitis (DS), an inflammatory process of the oral mucosa strongly associated with Candida spp and other microorganisms, as well as local and systemic factors. The impaired immune response against pathogens is among the inherent host factors that have been also associated with the pathogenesis of DS. Mononuclear phagocytes respond to the pathogens through phagocytosis followed by the production of several substances inside the phagosomes, among them are the reactive nitrogen species (RNS). A failure in these mechanisms may contribute to the DS development. Objective The aim of this study was to investigate the influence of aging on the internalization and the production of nitric oxide (NO) by peritoneal adherent cells (PAC), in response to Candida albicans (C. albicans). Material and methods PAC obtained from young and aged mice were challenged with dead or viable C. albicans by using predetermined proportions (cells:yeast) for 30 and 120 minutes. Phagocytosis was analyzed by acridine orange dye, and NO production by the Griess reaction. Results C. albicans phagocytosis by PAC from aged mice was similar to that of young mice, although the cells from older mice cells present more internalized fungi compared with matched control. In addition, a tendency towards impaired NO production by peritoneal mononuclear phagocytes from aged mice was observed. Conclusions PAC from aged mice may capture and store many fungi, which in turn may mean that these cells are effectively unable to eliminate fungi, probably due to impaired NO production. Therefore, considering the important role of C. albicans overgrowth in the pathogenesis of DS and the aspects observed in this study, aging may favor the onset and severity of local candidosis such as DS and its systemic forms.


Introduction
Candida-associated denture stomatitis (DS) is among the most common types of local candidiasis, an oral lesion present in more than 60% of the denturewearing individuals, with older persons being more frequently affected than the young population 14 . DS is strongly associated with the overgrowth of Candida spp on the inner denture surface, mainly consisting of Candida albicans (C. albicans), in addition to inadequate denture hygiene. Aspects inherent to the host are also strongly associated with DS, such immunosuppressive drugs and aging, since these hence to the establishment and maintenance of DS 14,25 .
C. albicans of the alimentary tract and mucocutaneous membranes of healthy humans. The host immune system is the major factor balancing the transition from commensalism to pathogenicity. When immune balance is disrupted, species of Candida are transformed into opportunistic pathogenic killers that can invade the tissues 7,23 . In this scenario, epithelial cells and mononuclear phagocytes, such as resident macrophages, provide the first line of defense against C. albicans by the initial fungal recognition and production of mediators that recruit and activate neutrophils. Besides, macrophages exert anti-Candida activities by means of phagocytosis and ability to kill this fungus by producing various enzymes and toxic substances in the phagosomes 1,23,30   Considering the phagocytosis of viable yeast, the greatest differences were observed at 30 minutes, since the proportion CaV2 at 120 minutes could not be counted. In the young group, the proportions CaV1 and Ca internalized fungi, varying between 79% and 87%.
PAC from aged group also showed high percentages of phagocytosis in the proportions CaV1 and CaV2, but As previously mentioned, the higher proportion of viable fungi:to cell (1:5 -CaV2) made it impossible to count the phagocytes at 120 minutes. In this period, we observed remnants of peritoneal mononuclear phagocytes with many budding yeasts and pseudohyphae, suggesting that the survival and proliferation mechanisms of C. albicans outweighed the defense of PAC that succumbed and died.
The quantitative evaluation of the number of dead or viable internalized yeasts (1,2,3,4, 5, or more) by PAC was also performed. In assays with dead fungi, the majority of peritoneal mononuclear phagocytes from the young group contained two yeasts within them, irrespective of the fungal proportions and the time of aged animals showed higher percentages of many minutes compared with the matched young animals. the proportion of CaD2 (Table 1).
Higher percentages of PAC with 1 or 2 viable internalized yeast/budding yeast/pseudohyphae were observed in the young group, except in the proportion of CaV2. Particularly in the proportion of CaV3, the percentage of PAC with one viable yeast/budding yeast/pseudohyphae within them were statically higher than those representing cells with 4, 5, or more internalized C. albicans analysis, no difference was observed in the assays with viable C. albicans and PAC from aged groups ( Table 1).
It is noteworthy that in the proportion of 1:5 (CaV2), after 120 minutes, PAC derived from both young and aged animals were covered by large amounts of budding yeasts and pseudohyphae of C. albicans. This high number of C. albicans associated Phagocytosis and nitric oxide production by peritoneal adherent cells in response to Candida albicans in aging: a collaboration to elucidate the pathogenesis of denture stomatitis with remnants of peritoneal mononuclear cells did not allow the cell counting, as previously reported.

Determination of nitric oxide (NO) production
In general, except in the proportions of low fungal burden, PAC from older animals showed a tendency to produce less NO than the cells from young animals.
This production was generally maintained over time.
The difference in NO production between aged proportions CaD1 ( Figure 3A) and CaV2 ( Figure 3B), The aforementioned trend was not observed in unstimulated cells, suggesting greater activation by PAC derived from young mice in trying to kill the fungi than those from aged mice.
A time-dependent increase in NO production was observed after PAC from young mice were stimulated with dead C. albicans (CaD1) (p=0.019). Furthermore, in NO production by PAC from young mice after 120 and 30 minutes of stimulation with dead and viable fungi, respectively (Figure 3).

Discussion
The role of innate immunity against infections in older persons is divergent. Some authors believe that the cells responsible for primary response are altered with aging 17,18,20,22,28 . Variable results were also obtained concerning the impact of aging on phagocytosis in mice. This function has been reported to be regulated negatively 18 , positively 17 , or even unchanged 22 with advancing age.
The present in vitro study with dead or viable C. albicans showed that PAC from aged animals exhibited a similar percentage of phagocytosis as that from However, in the group of older animals, after 120 minutes of challenge, the percentage of PAC with many was higher than those found in the young, regardless of the cell/yeast proportion or viability of the fungi.
Possibly, PAC from aged mice are storing these fungi that were successfully internalized due to their inability to kill and eliminate it. Furthermore, these cells could serve as disseminator vehicles, promoting dispersion of the pathogen in the body and protecting the fungus from other mechanisms of the innate immune response 3,30 .
In addition, we observed that cells from aged animals showed a tendency towards lower NO production than those of young animals. Altogether, these results suggested that the PAC from older animals may fail to kill internalized C. albicans, probably due to impaired NO production, since Elahi, et al. 8 (2001) showed the correlation between decreased NO production in saliva and increased viability of C. albicans the view that age is a contributing factor to functional impairment of innate immunity, representing a strong Candida diseases, as highlighted in numerous studies 17,18,20,22,28 . Particularly, advancing age has been considered one of the predisposing factors to Candida infections in denture wearers 12,13,16 .
Indeed, the development of candidiasis in humans appears to pose higher risk with advancing age 25 , he high number of budding yeasts and pseudohyphae of C. albicans or remnants of mononuclear cells did not allow cell counting. In addition, C. albicans itself presents the ability to escape from the microbicidal mechanisms of phagocytosis, including the potential to detoxify the phagosome, adapting to environments with microbicidal compounds, such as nitric oxide 6,10,11,27 .
It is possible that the suggested failure of PAC performance in aged animals, observed in the present study, resulted from mechanisms for subversion by the internalized fungus in the immune system.
In denture wearers, the C. albicans capacity of oral mucosa of these subjects to a continuous fungal load. Considering subjects with DS, the more common oral candidiasis, the communities found in dentures are usually formed by different Candida spp, mainly C. albicans, and various bacteria 4,16 . This association causes increased expression of virulence genes and those associated with epithelial damage and invasion 4 .
Following this line of reasoning, age is a really key aspect in the development and maintenance of DS. We also demonstrated impairment in the function of human salivary or blood neutrophils 12,13 and monocytes 24 obtained from volunteers with DS compared with denture wearers without this disease.