The immune response against parasitic infection is complex, and it involves many effectors and regulators components. Acanthamoeba spp. activate the classical TLR signaling pathway inducing NF-kB activation and increased secretion of inflammatory cytokines [25]. Induction of an inflammatory response by amoebas has been proposed as an important factor to determine the course of the parasite infection [8]. Nevertheless, little is known about the innate immune response induced by Acanthamoeba spp. in the kidneys and heart. To our knowledge, this is the first report showing TLRs expression in the kidneys and heart of hosts with disseminated acanthamoebiasis. Hence, further knowledge about the molecular and immunologic mechanisms induced by amoebas are important aspects to understand the course of infections and tissue invasion.
The occurrence of Acanthamoeba spp. infection has been described in patients following kidney transplantation [26–29], although in just one instance the amoebae were re-isolated from the kidney of patient with probable Acanthamoeba meningoencephalitis [30]. Despite the re-isolation of Acanthamoeba spp. from the mouse kidneys, the kidney profile performed in mouse serum indicated normal renal function [7]. Therefore, based on previous studies, the affinity of Acanthamoeba spp. to the kidneys can neither be confirmed nor excluded.
The factors involved in kidney damage and abnormal kidney function in parasitic diseases are still unknown. Based on studies on the role of Toll-like receptors in nephropathy induced by a Toxoplasma gondii infection, it was found that mainly TLR2 plays a role in kidney protection against T. gondii infection. Histopathological studies showed larger kidney damage in TLR2 deficient mice compared to those TLR4 deficient [31]. Studies on visceral leishmaniasis associated with renal abnormalities, suggest that Leishmania spp. antigens induce kidney inflammation by activating TLR2 and TLR4 receptors. The results indicate that kidney inflammatory processes and apoptosis involving TGF-β have a significant role in the pathomechanism of kidney damage in Leishmania donovani infection [32]. In the presented study, statistically significant differences in TLR2 expression occurred only between immunocompromised Acanthamoeba spp. infected mice and immunocompromised uninfected mice. We also noted differences in TLR4 expression between immunocompromised infected mice at 16 and 24 dpi and the control group, but the differences were not statistically significant (p = 0.06). Mun et al. [33] observed that TLR is not an essential molecule for protective immunity to low-dose of T. gondii cysts. In our study, the mice were infected with 10–20 thousands of amoebas, but Acanthamoeba spp. were re-isolated from 4 kidneys of immunosuppressed mice at 8 dpi, 6 kidneys at 16 dpi and 7 kidney’s mice at 24 dpi. Therefore, the higher expression of TLRs at 16 and 24 dpi may be a result of the low immunity level of the animals and/or the number of parasites that entered the kidneys.
Leemans et al. [34] have shown that TLR2 plays a critical role in the initiation of acute renal inflammation and early tubular injury. In histological preparations of kidneys of immunosuppressed mice, we found no inflammatory foci, but at the same we observed a lighter color of the kidney parenchyma [unpublished data]. In the present study, statistically significant changes in TLR2 expression were observed only at 24 dpi, the last day of the experiment. It is possible that histopathological changes in the form of inflammatory infiltrates could have been visible in the kidneys of mice in a longer-lasting acanthamoebiasis. In future studies, the experiment could be extended to 30–40 days post Acanthamoeba spp. infection and include determination of TGF-β expression, a cytokine which may mediate the progression of parasitic diseases in kidneys. Moreover, TGF-β induces transformation of kidney tubule cells to proliferating fibroblasts, causing fibrous changes in kidney parenchyma [35]. Such changes may be visible in histological preparations in the form of a lighter color of the kidney parenchyma.
Literature presents three cases of cardiovascular patients with acanthamoebiasis [6, 36, 37], although no parasite forms have been found in cardiac muscle cells [37]. Also, in this study no trophozoites or cysts were found in myocardial histological preparations from Acanthamoeba spp. infected mice, despite the fact that developmental forms of Acanthamoeba spp. were isolated from these samples. Therefore, it is not clear whether the amoebae were re-isolated from cardiac fragments or from residual blood in the heart, as the amoebae spread throughout the host organism via the bloodstream.
The Toll-like receptors expression has been reported in epithelium, endothelium and other cardiovascular cells [38, 39]. The results of studies conducted so far suggest that short-term activation of TLR receptors has a protective effect on the cardiovascular system, while long-term or excessive activation of these receptors induces chronic inflammation [38–40]. Cardiomyocytes in response to inflammatory stimuli are capable of secreting pro- and anti-inflammatory cytokines capable of initiating and regulating the inflammatory response, as well as chemokines, which recruit and activate appropriate inflammatory cells [41]. There is little data on TLR expression in the cardiac muscle cells of parasite-infected hosts. Ponce et al. [42] observed increased tlr2 gene expression in the heart of BALB/c neonatal mice infected with Trypanosoma cruzi. The authors suggested that these parasites may activate the host's innate immune response via different Toll-like receptors to protect cardiomyocytes from parasites. In contrast, Pereira et al. [43] suggest that a high TLR2 expression in patients with chronic Chagas cardiomyopathy may induce an increase in IL-1β, IL-12, and TNF-α, thereby elevating cardiac inflammation and contributing to heart dysfunction. Oliveira et al. [44] observed that a deficiency of TLR4 leads mice to being more susceptible to T. cruzi infection, as evidenced by a higher parasitemia and earlier mortality. However, it is important to point out that T. cruzi has an affinity for the heart, which is not the main biotope of Acanthamoeba spp. In this study, a statistically significant increase in TLR2 expression was seen in the heart of immunocompetent mice at 8 days post Acanthamoeba spp. infection compared to 24 dpi. When it comes to TLR4, statistically significant differences were found in the immunocompetent mice between 8 dpi and the uninfected mice, as well as at 8 dpi and 16 dpi. This shows that TLR2 and TLR4 induced an immune response at 8 days post Acanthamoeba spp. infection and thus protected cardiomyocytes from parasites, as confirmed by histological studies which did not show morphological changes [unpublished data]. Mogensen et al. [45] concluded that dexamethasone, an immunosuppressive drug, inhibits TLR-receptor signaling in Neisseria meningitidis and Streptococcus pneumoniae invasion. In our study, increased TLR4 expression in mice treated with the immunosuppressive drug at 16 days post Acanthamoeba spp. infection could be associated with delayed activation of this receptor by amoebae. In histological studies, mice treated with an immunosuppressive drug showed morphological changes in the form of hemorrhages and vacuolized cardiomyocytes with less acidic cytoplasm at 8 dpi and 16 dpi. In immunosuppressed mice at 24 dpi, we did not observe any morphological changes in the cardiac muscle [unpublished data].