In this study, the association of C. neoformans with glucose supplementation promoted pro-inflammatory and anti-inflammatory responses by increasing the IL-6, production and phospho-STAT3 activation, and decreasing the production of IL-8 and ERK1/2 phosphorylation. In addition, this combination demonstrated no cytotoxic effect but increased the cryptococcal internalization with reduction of percentage of fungi in the surface and its growth.
Diabetes patient’s present hyperglycemia which can cause directly or indirectly many complications in heart, kidneys and lungs [15]. The relationship between inflammation and hyperglycemia is widely studied. Hyperglycemia can display enhanced responsiveness and lead to inflammatory processes [16, 17]. Hyperglycemia can lead to changes in the functional properties of immune system cells, reprogramming them with expression genes of different profiles through epigenetic alteration. Furthermore, diabetes patients are also vulnerable to various infections[18–20] once that the hyperglycemia causes impaired granulocyte chemotaxis, phagocytosis and deficient cell-mediated immunity and may increase susceptibility to invasive fungal infections such as mucormycosis, candidiasis, coccidioidomycosis and cryptococcosis [7].
IL-6 is a pro-inflammatory cytokine, with pleiotropic roles in inflammation and infection[21]. In addition further studies. IL-6 is an important contributor to glucose and energy homeostasis. The definite role of IL-6 (beneficial or detrimental) on glucose metabolism is still debated [22]. In human immunodeficiency virus-positive patients with meningitis who present with cryptococcosis, a higher concentration of IL-6 in the cerebrospinal fluid has been associated with protective responses [23]. The production of IL-6 in human whole blood production was increased in the presence of capsule polysaccharides glucuronoxylomannan (GXM), galactoxylomannan (GalXM) and mannoprotein from C. neoformans [24]. Increased susceptibility to cryptococcal infection in IL-6 knockout mice infected with C. neoformans was also demonstrated when compared with wild-type mice [25]. Our results demonstrated association of both glucose supplementation and C. neoformans in BEAS-2B cells initiated overproduction of IL-6, an additive effect, compared to cells stimulated with separately pathogen. Therefore, increased production of IL-6 under the influence of elevated glucose levels may be more responsive to C. neoformans, thus leading to airway tissue damage and other diabetes-related complications. Furthermore, the overproduction of IL-6 due to the association between C. neoformans and glucose supplementation in bronchial epithelial cells may support host protection against cryptococcosis [26].
IL-6 increases the level of p-STAT3 remarkably [27]. STAT3 may be involved in the regulation of cellular inflammation [28]. Earlier results from our group demonstrated activation of STAT3 by C. neoformans in BEAS-2B cells [13, 29]. Hyperglycemia increases the phosphorylation of STAT3[30, 31] thereby contributing to the pathophysiology of tissue damage [32]. In a hyperglycemic model experimental it was showed that STAT3 was involved in the orientation of pulmonary disorders in diabetic rats [33]. Our results are consistent with these articles, as C. neoformans, induced STAT3 activation. Of note, the glucose supplementation in C. neoformans-infected cells showed an additive effect on STAT3 phosphorylation compared to isolated pathogen. These results are directly to the results found for IL-6. Therefore, the excessive induction of the IL-6/STAT3 signaling pathway due to the association of glucose supplementation with C. neoformans may be related to the control of cryptococcosis, but can also be correlated with an increase of airways damage caused by overreaction of inflammatory processes.
Extracellular signal-regulated kinase (ERK1/2) participates in the regulation of a large variety of processes, including cell adhesion, cell cycle progression, cell migration and others [34]. C. neoformans induced ERK1/2 activation in the NK-like cell line YT and primary peripheral blood NK cells [35]. However, GalXM, a cryptococcal polysaccharide from the capsule, reduced ERK1/2 activation in CD45 wild-type cells [36]. Earlier results from our group demonstrated activation of ERK1/2 by C. neoformans in BEAS-2B cells [37]. In hyperglycemic and STAT3 inhibitory conditions, ERK and STAT3 demonstrated an inverse relationship. Our results demonstrated that C. neoformans induced the phosphorylation of ERK1/2. In addition, downregulation of phospho-ERK1/2 activation was observed in cells treated with glucose supplementation and stimulated by C. neoformans compared to cells stimulated by C. neoformans. IL-8 is a chemoattractant mainly for neutrophils [38] and patients with diabetes display a marked elevation of circulating IL-8 levels [39]. However the modulation of IL-8 in the hyperglycemia depend on type of cells once its production did not change in smooth muscle cells, but increase in human umbilical venous endothelial cells (HUVEC) and aortic endothelial cells [40]. Earlier results from our group demonstrated increased IL-8 production by C. neoformans in BEAS-2B cells [13, 29, 41]. In our results, the glucose supplementation reduced the IL-8 production in C. neoformans-infected cells compared to cells only infected, which is associate to downregulation of ERK1/2, and it is independent of NF-κB. These results are in agreement with the study [42] in which Poly(I:C) alone, a TLR3 agonist, significantly induces expression of IL-8, however, the poly(I:C)-induced IL-8 expression was decreased by the addition of glucose (30mM). Using, inhibitors of IL-8 receptors it was observed a decreased morbidity in animal infected by airways with influenza virus and Streptococcus pneumoniae that was associated with decreased infiltration of neutrophils in the lungs [43]. Therefore, the downregulation of the IL-8/ERK1/2 signaling pathway caused by glucose supplementation in bronchial epithelial cells infected with C. neoformans could be beneficial reducing the pulmonary damage caused by exacerbation of inflammation but also could favor in fungal infection once IL-8 present pivotal role in the neutrophil activations.
NF-κB participate of inflammation and in the immune response against fungal infections [44]. Alveolar macrophages from mice infected with C. neoformans have increased NF-κB phosphorylation [45]. However, the NF-κB activity in lung epithelial cells could be entirely dispensable for the development of airway inflammation in response to to C. neoformans [41]. Earlier results from our group demonstrated activation of NF-κB by C. neoformans in BEAS-2B cells [13, 29]. The activation of NF-κB pathways by TNF-α and other cytokines are responsible for accumulation of macrophages in human diabetic nephropathy [46]. Our results demonstrated that C. neoformans increased the phosphorylation of NF-κB however no evident effect was observed by the addition of low and high glucose as in cells stimulated or not with C. neoformans.
No cytotoxic effect was observed in BEAS-2B cells stimulated by C. neoformans and/or glucose supplementation. However, in the MTT assay, an increase in the proliferative rate in the culture with C. neoformans was observed, which may be associated with addition of fungi, which can increase the production of formazan. Our results also demonstrated that hyperglycemia increased the internalization and consequently reduced the percentage of C. neoformans on the surface in BEAS-2B cells. In addition, the growth of C. neoformans was reduced by glucose supplementation in absence or presence of BEAS-2B cells. Fungal and the bacterial growth could be affected by glucose treatment. While bacteria rapidly react to an increased concentration of glucose, fungi appeared to only react to a minor degree to the addition, and with a different time dynamic [47–49].