In a run-down shack town, a fly drinks from a standing pool of water and unknowingly ingests unicellular protozoans of the genus Leishmania. As the sand fly takes its blood meal from an unsuspecting human, the parasites deploy into the fly’s saliva and enter the human’s bloodstream. There, they infect host macrophages and multiply until the cells fill and burst, which further spreads the parasite to more macrophages. In a short amount of time, the Leishmania parasite can induce painful, open lesions of the skin in what is known as cutaneous leishmaniasis. This disease is most prevalent in the poorest areas of the world and is due to a lack of quality housing, sanitation, and proper nutrition. And although the WHO and CDC report a global annual prevalence of approximately a million cases, the treatment options are surprisingly scarce; no vaccine is available and most drugs are ineffective against the parasite. Recent work from Ciara Gimblet, a CAMB-MVP student in the lab of Dr. Philip Scott, has unearthed a new aspect of the pathology of leishmaniasis that could be exploited to combat the disease.
Cutaneous leishmaniasis cases differ in severity of clinical outcomes. Earlier studies have found that the worst manifestation of this disease are a result of a heightened immune response and inflammation. Gimblet previously found that the cytokine Interleukin-22 (Il-22) mitigates cutaneous damage in leishmaniasis. Since Il-22 increases the production of antimicrobial compounds in the skin during infection, she hypothesized that the pathogenesis of cutaneous leishmaniasis is driven by alterations to the skin microbiome and induction of an inflammatory response.
To investigate how leishmaniasis impacts the skin microbiome, Gimblet et. al. first examined human patients with cutaneous Leishmania lesions. Taking swabs at the lesion, near the lesion, and contralateral to the lesion from each patient, the group found the lesions had lower microbial diversity than healthy skin and exhibited an unusually high percentage of Staphylococcus aureus or Streptococcus spp. While an increase in Staphylococcus and Streptococcus abundance is not unusual among skin inflammatory diseases, the patients in this study displayed a dramatic decrease in skin microbiome diversity. Furthermore, the microbiomes at and near the lesion were similar in diversity, consistent with the involvement of a local immune response..
Since it’s impossible to test human skin microbiome at the site prior to infection, the group used mouse models to elucidate the connection between cutaneous leishmaniasis and abnormal skin microbiome (also known as dysbiosis). C57BL/6 mice, a common strain used to study disease development and progression, were first swabbed on both ears before one ear was infected with Leishmania major. Swabs were then taken when lesion was fully developed at 6 weeks and when the lesion resolved at 12 weeks. Over the first 6 weeks, the microbiome at the infection site became less diverse and more dominated by Staphylococcus but returned to normal by 12 weeks.
If dysbiosis is a factor in leishmaniasis progression, then a high degree of dysbiosis would correlate with severe disease. To determine whether this was the case, Gimblet compared L. major pathogenesis in two mouse strains: C57BL/6 mice whose lesions heal by 12 weeks and BALB/c mice that develop chronic lesions. After 6 weeks, the BALB/c mice had lower microbial diversity and greater Streptococcus colonization than the C57BL/6 mice, thus strengthening Gimblet’s hypothesis. The group also created a mouse model of severe cutaneous leishmaniasis using C57BL/6 mice. They depleted Interleukin-12 (Il-12) because it was previously implicated separately in disease severity and in antimicrobial compound production. The IL-12-depleted mice phenocopied the BALB/c mice, again linking inflammation and dysbiosis to disease severity.
Gimblet et. al. identified a specific Staphylococcus species, S. xylosus, that is present on healthy mouse skin, but increases in abundance during cutaneous leishmaniasis in bacterial isolates taken from L. major infected mice. Previous studies had demonstrated that a change in even one species in skin microbiome can amplify inflammation. The group asked whether inoculating mice with S. xylosus could increase inflammatory response in the absence of L. major. Inflammation was seen only in mice infected intradermally with S. xylosus, as assessed by increased neutrophil recruitment and IL-1β expression. These results indicate S. xylosus contributes to inflammatory responses, but only in the presence of tissue damage.
Skin near Leishmania-infected lesions exhibits low microbial diversity (dominated by Streptococcus spp. and/or Staphylococcus aureus) when compared to healthy, contralateral skin. This phenomenon was observed in human patients and mouse models (A). Uninfected mice co-housed with Leishmania-infected mice develop dysbiosis. When challenged with pharmocologic irritants or L. major, the dysbiotic mice experience more tissue damage (B).
What’s more, Gimblet et. al. also found that skin bacteria can be transmitted from L. major infected mice to co-housed, uninfected mice without inducing an inflammatory response. This allowed the group to make a healthy mouse that has a skin microbiome similar to that induced by L. major infection. Using the dysbiotic model, they asked whether dysbiosis prior to infection causes more severe lesions and greater immune response. Prior to infection, dysbiotic mice had a heightened inflammatory response compared to mice with healthy skin. Moreover, dysbiotic mice infected with L. major developed more severe lesions and inflammation. Thus, an altered skin microbiome contributes to the severity of cutaneous leishmaniasis, in part due to an injury-induced inflammatory response. Whether dysbiosis of cutaneous leishmaniasis is transmitted between humans remains unknown and is a question that Gimblet hopes can be answered by sampling the skin of friends and family of affected individuals.
So how does leishmaniasis alter the proportions of different bacteria on skin? Gimblet postulates that the body attempts to fight L. major infection by expressing antimicrobial peptides (AMPs), which kill certain species of bacteria more effectively than others. Staphylococcus and Streptococcus are particularly resistant to AMPs and may dominate the microbiome while other bacteria are killed.
Since cutaneous leishmaniasis is exacerbated by Staphylococcus and Streptococcus, Gimblet hypothesized that antibiotics could be an effective treatment. Unfortunately, treating L. major-infected mice with sulfatrim and mupirocin, two antibiotics reported to treat cutaneous Staphylococcus infection, did not significantly change the microbiome or improve clinical outcome. The jury’s still out, given that antibiotics can also cause greater harm by disturbing the microbiome. Restoring the microbiome could be another promising treatment, so further studies might shed light on this hypothesis. Importantly, the study from Gimblet et. al. provides key insights into the mechanisms of leishmaniasis-induced dysbiosis, and opens doors for future research into the discovery of new therapeutic targets or treatment strategies to stop this insidious, microscopic parasite.
Ciara Gimblet, meanwhile, has set her sights on another family of tropical diseases, this time viral instead of protozoan. In her postdoc at the University of North Carolina, Chapel Hill, she is working on characterizing the immune response in adults and in fetuses during the course of infection with flaviviruses, including the infamous Zika virus.
Gimblet, C., Meisel, J.S., Loesche, M.A., Cole, S.D., Horwinski, J., Novais, F.O., Misic, A.M., Bradley, C.W., Beiting, D.P., Rankin, S.C., Carvalho, L.P., Carvalho, E.M., Scott, P., Grice, E.A. Cutaneous Leishmaniasis Induces a Transmissible Dysbiotic Skin Microbiota that Promotes Skin Inflammation. Cell Host Microbe 2017; 22(1):13-24.e4.