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African trypanosome parasites colonise the skin in a process important for parasite transmission. However, how the skin responses to trypanosome infection remain unresolved. Here, using a combination of spatial and single cell transcriptomics, coupled within vivogenetic models, we investigated the local immune response of the skin in both a murine model of infection. Our results provide several novel key findings previously unappreciated in the context of parasitic infections in the skin. First, we detected a significant expansion of a population of IL-17A-producing Vg6gdT cells in the infected murine skin compared to naïve controls that occur mainly in the subcutis, which we further validated at the protein level by flow cytometry. Second, interstitial preadipocytes located in the subcutis upregulate several genes involved in inflammatory signalling and antigen presentation, including T cell activation and survival.In silicocell-cell communication suggests that adipocytes triggergdT cell activation locallyvia Cd40, Il6, Il10,andTnfsf18signalling, amongst others. Third, mice deficient of IL-17A-producinggdT cells show extensive inflammation, increased frequency of dermal IFNg-producing CD8+T cells and limited subcutaneous adipose tissue wasting compared to wild-type infected controls, independent of TH1 CD4+T cells and parasite burden. Based on these observations, we proposed a model whereby adipocytes as well as Vg6gdT cells act concertedly in the subcutis to limit skin inflammation and tissue wasting. These studies shed light into the mechanisms ofgdT cell-mediated immunity in the skin in the context of African trypanosomes infection, as well as a potential role of immature and mature adipocytes as homeostatic regulators in the skin during chronic infection.&nbsp

Myotonic dystrophy type 1 is a complex disease caused by a genetically unstable CTG repeat expansion in the 3′-untranslated region of the DMPK gene. Age-dependent, tissue-specific somatic instability has confounded genotype–phenotype associations, but growing evidence suggests that it also contributes directly toward disease progression. Using a well-characterized clinical cohort of DM1 patients from Costa Rica, we quantified somatic instability in blood, buccal cells, skin and skeletal muscle. Whilst skeletal muscle showed the largest expansions, modal allele lengths in skin were also very large and frequently exceeded 2000 CTG repeats. Similarly, the degree of somatic expansion in blood, muscle and skin were associated with each other. Notably, we found that the degree of somatic expansion in skin was highly predictive of that in skeletal muscle. More importantly, we established that individuals whose repeat expanded more rapidly than expected in one tissue (after correction for progenitor allele length and age), also expanded more rapidly than expected in other tissues. We also provide evidence suggesting that individuals in whom the repeat expanded more rapidly than expected in skeletal muscle, have an earlier age-at-onset than expected (after correction for the progenitor allele length). Pyrosequencing analyses of the genomic DNA flanking the CTG repeat revealed that the degree of methylation in muscle was well predicted by the muscle modal allele length and age, but that neither methylation of the flanking DNA, nor levels of DMPK sense and anti-sense transcripts, could obviously explain individual- or tissue-specific patterns of somatic instability.

Background The diagnosis of gambiense human African trypanosomiasis (gHAT) typically involves 2 steps: a serological screen, followed by the detection of living trypanosome parasites in the blood or lymph node aspirate. Live parasites can, however, remain undetected in some seropositive individuals, who, we hypothesize, are infected with Trypanosoma brucei gambiense parasites in their extravascular dermis. Methods To test this hypothesis, we conducted a prospective observational cohort study in the gHAT focus of Forecariah, Republic of Guinea. Of the 5417 subjects serologically screened for gHAT, 66 were enrolled into our study and underwent a dermatological examination. At enrollment, 11 seronegative, 8 unconfirmed seropositive, and 18 confirmed seropositive individuals had blood samples and skin biopsies taken and examined for trypanosomes by molecular and immunohistological methods. Results In seropositive individuals, dermatological symptoms were significantly more frequent, relative to seronegative controls. T.b. gambiense parasites were present in the blood of all confirmed cases (n = 18) but not in unconfirmed seropositive individuals (n = 8). However, T. brucei parasites were detected in the extravascular dermis of all unconfirmed seropositive individuals and all confirmed cases. Skin biopsies of all treated cases and most seropositive untreated individuals progressively became negative for trypanosomes 6 and 20 months later. Conclusions Our results highlight the skin as a potential reservoir for African trypanosomes, with implications for our understanding of this disease’s epidemiology in the context of its planned elimination and underlining the skin as a novel target for gHAT diagnostics., Live trypanosomes can remain undetected in the blood of individuals seropositive for sleeping sickness. Here, we show that they could be infected with parasites in their extravascular dermis, highlighting the skin as a potential reservoir for trypanosomes.

posté sur MedRxiv le 25 février 2020 : https://www.medrxiv.org/content/10.1101/2020.02.24.20026211v1; International audience; Background The diagnosis of Human African Trypanosomiasis (HAT) typically involves two steps: a serological screen, followed by the detection of living trypanosome parasites in the blood or lymph node aspirate. Live parasites can, however, remain undetected in some seropositive individuals, who we hypothesize are infected with Trypanosoma brucei gambiense parasites in their extravascular dermis. Methods and findings To test this hypothesis, we conducted a prospective observational cohort study in the gambiense HAT (gHAT) focus of Forecariah, in the Republic of Guinea. 5,417 subjects in this disease foci underwent serological screening for gHAT. Of these individuals, 66 were enrolled into our study, of whom 40 were seronegative, 8 were seropositive but unconfirmed, and 18 confirmed gHAT cases. Enrolled individuals underwent a dermatological examination, and had blood samples and skin biopsies taken and examined for trypanosomes by molecular and immuno-histological methods. In confirmed cases, dermatological symptoms were significantly more frequent, relative to seronegative controls. T. b. gambiense parasites were present in the blood of all confirmed cases but not in unconfirmed seropositive individuals. However, trypanosomes were detected in the dermis of all unconfirmed seropositive individuals and confirmed cases. After 6 and 20 months of treatment, dermal trypanosome numbers in skin biopsies of confirmed cases progressively reduced. Conclusions Our results thus highlight the skin as a potential reservoir for trypanosomes, with implications for our understanding of this disease's epidemiology in the context of its planned elimination and highlighting the skin as a novel target for gHAT diagnostics.

Infection by Trypanosoma brucei gambiense is characterized by a wide array of clinical outcomes, ranging from asymptomatic to acute disease and even spontaneous cure. In this study, we investigated the association between macrophage migrating inhibitory factor (MIF), an important pro-inflammatory cytokine that plays a central role in both innate and acquired immunity, and disease outcome during T. b. gambiense infection. A comparative expression analysis of patients, individuals with latent infection and controls found that MIF had significantly higher expression in patients (n = 141; 1.25 ± 0.07; p

Myotonic dystrophy type 1 (DM1) is a multisystem disorder, caused by expansion of a CTG trinucleotide repeat in the 3′-untranslated region of the DMPK gene. The repeat expansion is somatically unstable and tends to increase in length with time, contributing to disease progression. In some individuals, the repeat array is interrupted by variant repeats such as CCG and CGG, stabilising the expansion and often leading to milder symptoms. We have characterised three families, each including one person with variant repeats that had arisen de novo on paternal transmission of the repeat expansion. Two individuals were identified for screening due to an unusual result in the laboratory diagnostic test, and the third due to exceptionally mild symptoms. The presence of variant repeats in all three expanded alleles was confirmed by restriction digestion of small pool PCR products, and allele structures were determined by PacBio sequencing. Each was different, but all contained CCG repeats close to the 3′-end of the repeat expansion. All other family members had inherited pure CTG repeats. The variant repeat-containing alleles were more stable in the blood than pure alleles of similar length, which may in part account for the mild symptoms observed in all three individuals. This emphasises the importance of somatic instability as a disease mechanism in DM1. Further, since patients with variant repeats may have unusually mild symptoms, identification of these individuals has important implications for genetic counselling and for patient stratification in DM1 clinical trials.

Trypanosoma brucei is the causative agent of African sleeping sickness in humans and one of several pathogens that cause the related veterinary disease Nagana. A complex co-evolution has occurred between these parasites and primates that led to the emergence of trypanosome-specific defences and counter-measures. The first line of defence in humans and several other catarrhine primates is the trypanolytic protein apolipoprotein-L1 (APOL1) found within two serum protein complexes, trypanosome lytic factor 1 and 2 (TLF-1 and TLF-2). Two sub-species of T. Brucei have evolved specific mechanisms to overcome this innate resistance, Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. In T. b. Rhodesiense, the presence of the serum resistance associated (SRA) gene, a truncated variable surface glycoprotein (VSG), is sufficient to confer resistance to lysis. The resistance mechanism of T. b. Gambiense is more complex, involving multiple components: reduction in binding affinity of a receptor for TLF, increased cysteine protease activity and the presence of the truncated VSG, T. b. Gambiense-specific glycoprotein (TgsGP). In a striking example of co-evolution, evidence is emerging that primates are responding to challenge by T. b. Gambiense and T. b. Rhodesiense, with several populations of humans and primates displaying resistance to infection by these two sub-species.

Humans are protected against infection from most African trypanosomes by lipoprotein complexes present in serum that contain the trypanolytic pore-forming protein, Apolipoprotein L1 (APOL1). The human-infective trypanosomes, Trypanosoma brucei rhodesiense in East Africa and T. b. gambiense in West Africa have separately evolved mechanisms that allow them to resist APOL1-mediated lysis and cause human African trypanosomiasis, or sleeping sickness, in man. Recently, APOL1 variants were identified from a subset of Old World monkeys, that are able to lyse East African T. b. rhodesiense, by virtue of C-terminal polymorphisms in the APOL1 protein that hinder that parasite’s resistance mechanism. Such variants have been proposed as candidates for developing therapeutic alternatives to the unsatisfactory anti-trypanosomal drugs currently in use. Here we demonstrate the in vitro lytic ability of serum and purified recombinant protein of an APOL1 ortholog from the West African Guinea baboon (Papio papio), which is able to lyse examples of all sub-species of T. brucei including T. b. gambiense group 1 parasites, the most common agent of human African trypanosomiasis. The identification of a variant of APOL1 with trypanolytic ability for both human-infective T. brucei sub-species could be a candidate for universal APOL1-based therapeutic strategies, targeted against all pathogenic African trypanosomes., Author Summary African trypanosomes are protozoan parasites that affect both humans and animals in poor rural areas of sub-Saharan Africa, and are a major constraint on health and agricultural development. Disease control is principally dependent on the administration of drugs, which are old and largely unsatisfactory. Humans are naturally resistant to infection by most African trypanosomes species because of a lytic protein component in their blood, called APOL1. However, human-infective trypanosomes, T. b. rhodesiense in East Africa, and T. b. gambiense in West Africa, have evolved separate mechanisms to disarm this lytic protein and cause disease. Recently, variants of APOL1 were discovered in some primates that are able to kill the East African human disease-causing sub-species. These APOL1 variants form the basis of current attempts to create novel therapeutic interventions that can kill both animal and human-infective trypanosomes. In this study, we show that another variant of the same protein from a West African baboon species is able to kill, not only East African human-infective trypanosomes, but also the West African parasites, which causes the majority of human African trypanosomiasis cases. This new APOL1 variant could be a potential candidate for anti-trypanosomal therapies targeted at all pathogenic trypanosome species.

Trypanosoma brucei is a protozoan parasite of major public health and economic importance in sub-Saharan Africa, where it is the causative agent of sleeping sickness in man and Nagana in cattle. The complete genome sequence of T.brucei is now available and the diploid genetic system has recently been demonstrated to be Mendelian. This opens up the possibility of using a classical genetic approach to identify genetic loci that determine important phenotypic traits in this parasite, such as host specificity, drug resistance, and pathogenicity. A genetic map of the non human-infective subspecies, T.b.brucei, has already been assembled and successfully used in quantitative trait analysis of a number of traits specific to this pathogen. This thesis describes the construction of a separate genetic map for the sub-species responsible for > 90% of human African trypanosomiasis infections, T.b.gambiense, which differs significantly from T.b.brucei in many key phenotypes. The genetic linkage map was constructed from the analysis of 119 polymorphic microsatellite markers in a population of 38 F1 progeny, obtained from the genetic cross of a T.b.gambiense group 2 strain, STIB 386, with a T.b.brucei strain, STIB 247. Eleven major linkage groups were resolved, one for each of the megabase chromosomes, resulting in a total genetic map length of 733 cM, and an average map unit size of 24 Kb/cM. The map provides a 90% probability of a marker being within 268 Kb of any genetic locus. A comparative analysis of the T.b.gambiense and T.b.brucei genetic maps revealed synteny and marker order to be conserved between the two sub-species. However, variation was observed in the location of regions of high and low recombination frequency (hot and cold spots) in the two maps. The genetic linkage map presented here is the first available for T.b.gambiense and can now be utilised to find the location within the genome of genes responsible for phenotypic traits in this clinically important sub-species. These traits include human infectivity, tsetse transmissibility and virulence, in addition to sensitivity to the trypanocidal drug, pentamidine, for which phenotypic variation between the parents was characterised both in vitro and in vivo in this thesis. The ability of the T.brucei genetic maps to pinpoint loci underlying phenotypic variation is limited by the number of recombination events, and therefore progeny, available for analysis. To increase the utility of this approach for future studies, an improved method for progeny isolation from uncloned genetic cross populations was also developed. This in vitro bloodstream cloning procedure is scalable and efficient, and replaces a time consuming and technically demanding in vivo method. Twelve new progeny clones were isolated by this approach during the trial and incorporated into the analysis, representing a step toward a higher resolution second-generation genetic map. Finally, whilst undertaking genotyping analysis with microsatellite markers the development of spontaneous chromosome 10 abnormalities was observed. A detailed investigation identified seven laboratory-adapted T.brucei lines in which loss of heterozygosity appeared to have occurred. These alterations to the karyotype significantly exceeded the well-characterised genomic rearrangements of subtelomeric regions that are frequently associated with antigenic variation in African trypanosomes. Microsatellite analysis, pulsed field gel electrophoresis and Illumina next generation sequencing demonstrated these changes to be the product of mitotic recombination events in the chromosome core, resulting in an extensive loss of heterozygosity of up to 75% of the chromosome and correlated with an improved growth phenotype. Further work is now required to determine the extent and frequency with which these abnormalities might occur, however these findings do highlight the potential instability of the molecular karyotype of T.brucei in prolonged in vitro culture.