Leishmania spp. exhibit obligate intracellular parasitism within phagocytic cells of their vertebrate hosts, particularly macrophages (Alexander et al., 1999). The mechanism by which this genus is able to successfully parasitise macrophages has been of interest to the scientific community both for its novelty and medical relevance. Leishmania parasites, regardless of species, are able to enter these antigen-presenting cells that are central to cell-mediated immunity without getting recognized and killed. In order to keep the infections incubating within the host, these parasites and their vector downregulate immune functions significantly starting from the moment of entry, moving forward years into the initial infection. However, the gap in knowledge as to the extent to which regulating cell-mediated immune functioning is associated with parasite-specific pathogenesis remains unclear. Leishmania infections involve purinergic signalling as the basis of the host’s response. Dendritic cells present metacyclic-promastigotes to naive T-cells, which then develop into TH-1 cells and secrete interferon-gamma. This presentation results in macrophage secretion of nitric oxide that kill some of the host cells. The dead host cells contain high amounts of ATP and nucleotides that feedback to inhibit the process from continuing and killing more parasitized cells (Gregory et al., 2008). The immunological basis of Leishmaniasis infections are of significant interest to researchers because of the medical importance of the different presentations of this disease. There are up to 1,600,000 new cases a year of Leishmaniasis, where 70% of the cases are found in poverty-stricken regions, 90% of which come from the Indian subcontinent, Brazil, and Sudan (Singh et al., 2010; Moore and Lockwood, 2010). Current treatment methods for the various manifestations of this disease are problematic with respect to their efficacy and cost. Disease treatment and presentation is largely based on the individual, and therefore therapy must be closely monitored on a case-by-case basis. This becomes difficult in the aforementioned regions, where it is not feasible for patients to have the time and money necessary to properly treat the parasite (Moore and Lockwood, 2010). While this scenario is common-place for many parasitic infections, it is highlighted during Leishmania infections, which have extremely different manifestations of the disease depending on which species within the genus is causing the infection. The three manifestations of the Leishmaniasis are Visceral, Cutaneous, and Mucosal; each of which is caused by different species. Visceral Leishmaniasis, the most deadly form, presents with hepatosplenomegaly, fever, weight loss; it results in 100% mortality rate if left untreated within the first two years of infection. Cutaneous Leishmaniasis is characterized by painless and localized, ulcerative skin lesions that last several months. Mucosal Leishmaniasis can be its own its own infection or result from untreated Cutaneous Leishmaniasis; it presents with a degradation of the mucous membranes (Aoun and Bouratbine, 2014). Despite parasites within the genus Leishmania exhibiting almost identical life cycles and morphologies, they demonstrate the extremely different pathologies. This leads to the scientific rationale for in this review in which the etiology of these differences in pathogenesis is determined. Since macrophages are key to the development of the parasite into its infectious form, do Leishmania spp use different mechanisms to alter them and other cells of the immune system? The topic at hand reviews the immunological basis for how Leishmania major, Leishmania donovani, and Leishmania braziliensis cause Cutaneous, Visceral, and Mucosal Leishmaniasis respectively. In essence, the paper will determine whether or not there is a significant difference in the mechanisms that Leishmania major, Leishmania donovani, and Leishmania braziliensis utilize to infect immune system cells and cause their varying pathologies.Comparison and ContrastSimilarities in Life Cycle and Morphology There are highly conserved characteristics present within the genus Leishmania. With respect to its morphology, Leishmania spp is a trypanosomal parasite. It has one flagella, whose size depends on the stage of infection; and a kinetoplast disk that contains its DNA (Gregory et al., 2008). There are 20 species of Leishmania that are spread by 30 species of Phlebotomus and Lutzomyia sandfly across 90 countries (Bañuls et al., 2007). In addition to being intermediate host, the sandfly plays a role in the vertebrate hosts’ initial immunological response by secreting a salivary peptide upon taking a blood meal which is associated with the inhibition of apoptotic factors such as tumor-necrosis-? and nitric oxide, along with quicker lesion formation (Hall and Titus, 1995). Through the proboscis, the host is inoculated with metacyclic promastigotes that are the non-infectious stage of Leishmania. This form is flagellated and coated with glycoconjugates and receptors that allow the parasite to survive in the sandfly gut. These proteins also prevent the activation of the complement immune cascade, and create ease of access into the macrophage cells (Alexander et al., 1999). The life cycle of Leishmania spp is identical, regardless of which species is infecting the host. Upon infection with promastigotes, the opsonization of two surface receptors occurs where they become metacyclic, and this process also allows for entry into the macrophages without activating cell-mediated immunity (Alexander et al., 1999). When phagocytosed, the promastigotes transforms into amastigotes and are able to multiply and spread in the macrophages, potentially spreading to other organs. When a sandfly takes another blood meal of an infected individual, the amastigotes transform to procyclic promastigotes (See Figure 1), where they can actively divide by binary fission. The longer flagella that is present in promastigotes facilitates the attachment of this stage of the parasite to the midgut of the sandfly (Killick-Kendrick, 1990), where it is protected from hydrolytic stomach enzymes by glycoproteins (Alexander et al., 1999). Despite these similarities in life cycle, parasite and vector morphology, significant data exists suggesting differences in the immunological basis of these varying parasitic infections; much of which centering around changes to host inflammatory responses and macrophage gene expression. Immunological Basis of L. major Immune Attack Leishmania major, the cause of Cutaneous Leishmaniasis, demonstrates novel properties with respect to altering the host’s immunological response to a parasitic infection. The way in which this parasite alters the host’s inflammatory responses are unique when compared to other species within the same genus. One way in which it does so is by inducing an inflammatory response that attacks T-helper cells, and weakening the immune system so that the infection can spread throughout the skin with less antigen-presenting macrophages (De Freitas et al., 1999). Different genes are shut off during L. major expression when compared to L. donovani. In particular, 26 genes, related to E1 prostaglandins, which cause inflammation in response to infection, are synthesized less during Leishmania major infections. Research suggest that this may be a factor in the fact this manifestation of the parasite is less severe, resulting in localized skin lesions that are more treatable (Gregory et al., 2008). The same study found that mutations in Cox2, Tdag51, and Phlda-1, occur during Leishmania major infections that have roles in down-regulation of immune cell functioning as well. Further research suggests that Leishmania major infections are associated with altering the expression of interleukin-10, the consequences of this having implications in why the parasite is able to evade host immune responses. Interleukin-10 is known as an anti-inflammatory immunomodulator. The protein inhibits TH-1 cytokines and other molecules on the surface of macrophages. The inactivation of interleukin-10 by promastigotes of Leishmania major results in an altered ratio of CD4(+)CD25(+) effector cells. These immune cells would otherwise accumulate at the surface of the skin during an infection, but during Cutaneous Leishmaniasis they are disrupted, which is suggested to be a reason why infecting the surface of the skin may be beneficial for this species (Belkaid et al., 2002). Treatment of this species of Leishmania with mesenchymal stem cells rich in lipopolysaccharides that had the parasite antigen were able to counter this particular mechanism of attack by increasing interleukin-10 production  (Khosrowpour et al., 2017). Perhaps the most novel property present only within the species Leishmania major is the presence of a protein called Leishmania major phosphatase, LmPRL-1, that is excreted via an exocytotic pathway. It was identified by comparing its amino acid groups, demonstrated in Figure 2, through electron microscopy and phylogenetically linked to human phosphatase. This protein is a homologue for mammalian phosphatase, presenting with a similar structure and regulatory role in cell development and plasticity.  The phosphatase is more prevalent in infections where the parasite load is high, and it is secreted at a constant steady rate with the purpose of helping this species survive in macrophages and become increasingly infectious (Leitherer et al., 2017). Figure 3 demonstrates empirically that the presence of this protein in promastigotes are associated with increasing infectivity and survival. The presence of this protein provides evidence that there is a difference between the immunological basis for how these parasites within the same genus are able to result in such varied expressions of Leishmaniasis. Immunological Basis of L. donovani Immune Attack Leishmania donovani results in Visceral Leishmaniasis, the expression of the disease that has the highest mortality rate when compared to its cutaneous and mucocutaneous counterparts (Moore and Lockwood, 2010). Leishmania donovani infections have been found more in areas with higher incidence of malnutrition, which causes a decreased interleukin-10 as part of the body’s natural response to starvation. There was a strong positive correlation between the level of starvation present in promastigote-infected mice and the level of infectivity measured with respect to how many cells were parasitized and the extent of the infection spreading to other organs (Anstead et al., 2001). While the increase in interleukin may be compared to mice infected by Leishmania major, Leishmania donovani infections were also shown to increase prostaglandin production, which could result in the downregulation of inflammatory responses. Consequently, most of the research in the field correlates infection with Visceral Leishmaniasis and the downregulation of macrophage gene expression. While this is unique from manifestations of Cutaneous Leishmaniasis mentioned previously, it is similar in the sense that the genes altered are involved in inflammatory responses similar to the ones that occur during Leishmania major. They only differ in the exact mechanism by which they are changed. The most pronounced of these differences is the 90 genes that are suppressed two-fold during Leishmania donovani infections (See Figure 4), a 37% reduction when compared to the total number of genes present in a macrophage (Gregory et al., 2008). The same study attempted to address the question at hand by comparing what factors within the host’s’ immune response are the reason that Leishmania major leads to Cutaneous Leishmaniasis, while Leishmania donovani leads to Visceral Leishmaniasis. Polymerase Chain Reaction of blood samples of individuals with manifestations of both diseases was run, and it was demonstrated that both diseases altered PGE2, CoX2, and metallothionein. PGE2 is associated with suppression of TH-1 immune cells, while CoX2 is the rate limiting step in the production of prostaglandin, and metallothionein protects cells from oxidative stress. Figure 5 demonstrates that while similar genes were repressed by both infections, Leishmania donovani did so significantly more than Leishmania major (Gregory et al., 2008). This suggests that the differences within how the diseases manifest may be less a question of “how?,” but “how much?,” since the mechanism for altering the immune system is the same. The differences are suggested to arise because of the extent to which the genes are suppressed. A similar concept was seen in the aforementioned study comparing the visceralization of Leishmania donovani amongst different levels of starving mice, and the more deadly manifestations of the parasite were the result of the more starved mice (Anstead et al., 2001). Another way in which the mechanisms by which Leishmania donovani and Leishmania major alter the immune system in a similar way is through changing the level of genes pivotal to macrophage accessory functions involving TH-1 cells. In the case of Leishmania donovani, interferon-gamma production resulted in TH-1 expression increasing. Conversely, interleukin-2 decreased which resulted in a decrease in TH-2 in infected cells (Manamperi et al., 2017). A similar concept what was seen in the aforementioned Leishmania major study, where silencing interleukin-10 production caused the suppression in TH-1 cells (Belkaid et al., 2002). While both species within the genus are resulting in a similar pattern of suppressing TH-1 and cell-mediated immunity, it is evident that they are reaching this through different mechanisms. Visceralization of Leishmaniasis is also associated with silencing of several genes involved in cell-mediated immunity that have been found to be produced by macrophages, including major histocompatibility proteins, WNT5A, and programmed death-1 receptors. The expression of major histocompatibility complex I and II was found to be suppressed in Leishmania donovani infections. In doing so, the parasite is able to prevent the activation of T-lymphocytes. The extent of the suppression was found to be associated with the intensity of visceralization seen in infected mice. However, the parasite was found to be better adapted to silence genes within MHC II (Reiner et al., 1987). Another aspect of the immune system that is silenced during Leishmania donovani infections is the WNT5A gene that activates the WNT5A signalling pathway involved in inflammatory response, where the extent of WNT5A silencing could be positively correlated to the level of visceralization and infectivity (Chakraborty et al., 2017). Additionally, the downregulation of programmed death-1 receptors in this species of Leishmania was shown to prevent the activation of protein kinase B, which would otherwise induce apoptosis of infected cells (Roy et al., 2017).Immunological Basis of L. braziliensis Immune Attack Leishmania braziliensis is the cause of Mucosal Leishmaniasis, which can result in extensive damage to the soft palate and breathing complications (Aoun and Bouratbine, 2014). This parasite and its resulting disease is unique because it may either be the result of untreated or poorly-treated cutaneous lesions, or a completely separate manifestation of the disease. In essence, Leishmania braziliensis may cause Cutaneous or Mucosal Leishmaniasis. The varying pathologies are what make this particular species of particular interest to researchers. Understanding what triggers the different expressions of the disease has implications in the immunological basis for the differences in pathologies seen in the three different diseases associated with Leishmaniasis. A novel property seen within this species of Leishmania that may be associated with its unique pathology is the elevated expression of AIM2. The AIM2 gene was found in more severe cases of Leishmania braziliensis, particularly those that cause its mucosal manifestation. The same study also found that the expression of AIM2 mRNA resulted in cases of the disease that were more difficult to treat and had lowered efficacy of antileishmanial medication (Moreira et al., 2017). However, AIM2 is not the only protein that is differentially expressed in the two possible forms of Leishmania braziliensis. Analysis of mice with the inflammatory protein Annexin A1 indicated that some of the differences in pathology seen may be due to higher Annexin A1 expression in Mucosal Leishmaniasis. The cutaneous form of the disease also had altered inflammatory proteins, but to an extent that was more manageable by the host (Oliveira et al., 2017). The three manifestations of Leishmaniasis discussed demonstrate a similar pattern, where the same genes are altered to different extents; suggesting that the severity of the disease’s expression is partially the result of differential expression of immune cell associated genes. When the expression of genes within the immune system were analyzed in dendritic cell monocytes infected with either Visceral or Mucosal Leishmaniasis, they were both found to upregulate CD86+ and downregulate CD209+ to different extents that did not reach statistical significance. However, when their expression of tumor necrosis factor, which is compared in Figure 6, showed a significant difference in the expression between the parasite species, with only Leishmania braziliensis upregulating the gene. Leishmania braziliensis infected hosts are therefore still able to induce apoptosis of the parasitized cells, while Visceral Leishmaniasis infected individuals’ cells are not apoptosed and are able to spread to other organs (Falcão et al., 2017).DiscussionParasite species within the genus Leishmania exhibit very different pathologies despite similarities within their morphologies and life cycles. Based on the literature reviewed, several patterns may be observed within the mechanisms that parasites of the genus use to cause their differing manifestations of Leishmaniasis. Immune cells that are actively expressed by macrophages exhibit changes to the expression of several genes, especially with respect to those associated with inflammatory responses such as AIM2 and interferon-gamma. Therefore, the basis of the observed patterns is the reversal of immune cell functioning by a series of down-regulations to the benefit of the parasite. Additionally, the severity of these three diseases has been empirically demonstrated to be dependent on the extent of gene expression. Not only is the host’s response dynamic depending on what species it is infected with, but the response of immune cells is also significantly dynamic. Because so many genes within the immune system are changing in response to the parasite, there is no singular factor evident yet that determines pathogenesis. Despite there being a general pattern amongst Leishmania spp infections, research suggests the parasites use different mechanisms to result in the changes to immune cell functioning. For example, while Leishmania donovani and Leishmania major both result in the upregulation of TH-1 immune cells, Leishmania major does so by the downregulation of interleukin-10, while Leishmania donovani does so by interferon-gamma production. Consequently, infection by a parasite of the genus Leishmania is associated with differences in the expression of CoX2, PGE, and metallothionein, regardless of the species. However, visceralization of the disease is associated with significantly lowered expression of these genes when compared to its mucosal counterpart. A similar concept by confirmed in PCR analysis of the 245 genes expressed by macrophages during Leishmania infection, which demonstrated a difference in the expression of 26 genes between Visceral and Cutaneous Leishmaniasis. These differences in the mechanisms used to alter the expression of immune cells is associated with their differences in pathogenicity, as the severity of the disease is contingent upon the extent of the change to these genes. This is also evident in the fact that when comparing Visceral and Mucosal Leishmaniasis, only the latter results in apoptosis as the result of differential expression of tumor necrosis factor alpha. Due to these differences in the mechanism by which cells of the immune system are altered by Leishmania spp, and the association of these differences with their varying pathologies, the null hypothesis presented is rejected. Therefore, Leishmania major, Leishmania donovani, and Leishmania braziliensis utilize different mechanisms to infect immune system cells and cause their varying pathologies. These species use a combination of activating and inhibiting certain cells so that they are effective in maintaining their infection in the host, often for years following initial infection. The data presented suggests that immune responses are species-specific and and depend on differential inflammatory responses from macrophage, lymphocyte, and cytokine activity.The broader implications of understanding the immunological basis for the Leishmaniasis pathologies with respect to devising affordable treatment methods for these diseases with a better response rate and efficacy. For example, understanding that Cutaneous Leishmaniasis results in the upregulation of TH-1 through lowered prostaglandin synthesis led to creating a treatment method with indomethacin to counteract this mechanism and significantly lower the parasite load (De Freitas et al, 1999). Pretreating mesenchymal stem cells with a parasite antigen was also successful in removing the interleukin-10 deficit created by Leishmania major infections (Khosrowpour et al., 2017). Additionally, WNT5A introduction reduced the intensity of the parasitic load of Leishmania donovani infections by counteracting the mechanism by which the parasite lowers WNT5A expression (Reiner et al., 1987). However, there are several limitations to the data presented amongst the literature reviewed. Amongst these is the fact that all of the data was conducted on in vitro studies of cell cultures instead of in vivo analysis, which would provide a better understanding of how the parasite is working within the vertebrate host’s body. Additionally, the studies were all conducted in short term and do not demonstrate the effects of the parasite at a later stage of infection. A limitation discussed by some of the researchers is the fact that the studies were conducted on promastigote cultures because of their ease of being recreated in vitro, while the infectious form of the disease is the the amastigote. Further research must be conducted using on the actual role of the macrophage genes expressed now that they have been identified. Conducting amastigote studies over a longer span of time might reveal more about how the parasite acts during its infectious stage at different points in its life cycle. Because there are different external aspects that affect the presentation of any disease, ruling out environmental and epidemiological factors in the expression of the different Leishmaniasis pathologies should also be considered in further research conducted.

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