Genotypic Differences among Isolates of L. Braziliensis and Host Factors in The Pathogenesis of Disseminated Leishmaniasis

Disseminated Leishmaniasis (DL) is an emerging and severe form of Leishmania braziliensis infection dened by the presence of 10 and up to more than 1,000 skin lesions. The mechanisms underlying parasite dissemination remain unknown. Genotypic differences among species of L. braziliensis have been associated with different clinical forms of disease. The present work compared the function of monocytes obtained from cutaneous leishmaniasis (CL) and DL patients in response to infection with isolates of L. braziliensis pertaining to both of these two clinical forms of disease. Mononuclear cells obtained from DL and CL patients were infected with different L. braziliensis isolates. Numbers of infected cells, parasite load, respiratory burst, TLR2 and TLR4 expression and cytokine production were evaluated. DL isolates infected more monocytes, induced greater respiratory burst, higher expression of TLR2 and TLR4 and more cytokine production compared to isolates from CL patients regardless of the origin of monocytes used (DL or CL). However, greater parasite multiplication and higher TLR expression were seen in monocytes from DL patients compared to CL following infection with DL isolates. Our results indicate the participation of both parasite genotype and host factors in the pathogenesis of DL. ,


Introduction
Disseminated leishmaniasis (DL), a severe form of Leishmania (Viannia) braziliensis infection, is de ned by the presence of more than 10, and up to 1,000 papular, acneiform and ulcerated lesions across at least two separate parts of the body. 1 DL has also been documented in infection by other Leishmania species 2,3,4,5 , and differs from classical cutaneous leishmaniasis (CL), which is characterized by the presence of a single or few-well delimited ulcers with raised borders. Often confused with diffuse CL (DCL), DL is considered clinically, immunologically and histopathologically distinct from DCL 6,7,8,9 . While DCL patients exhibit poor lymphocyte proliferation and cellular production of IFN-γ upon exposure to soluble leishmania antigen (SLA) 8,9 an impaired Th1 immune response has not been documented in DL 10 . In fact, cytokine expression at the lesion site is similar between DL and CL, and no comparative histopathological or immunochemical differences have been evidenced on ulcer tissue analysis 10,11 .
Recently, emphasis has been placed on the role of CD8 + T cells in the pathogenesis of L. braziliensis infection. CD8 + T cells from CL patients were observed to kill leishmania-infected monocytes, yet did not kill parasites 12 . Moreover, transcriptomic analysis of lesions from CL patients revealed the high expression of genes associated with the cytolytic pathway, and CD8 (+) T cells obtained from lesions exhibited a cytolytic phenotype 13 . Moreover, disease progression and metastasis in L. braziliensisinfected mice was found to occur independently from parasite burden, instead being directly associated with the presence of CD8 (+) T cells 13 .
In spite of their participation in disease pathology, macrophages are the main cell type responsible for leishmania killing 14,15 . Regarding monocytes, also known to participate in the host in ammatory response in CL, the enhancement of intermediate monocytes is notable, as these are the most important source of TNF, a cytokine associated with tissue damage in TL 16 .
The Toll-like receptor (TLR) signaling pathway is a primary defense mechanism against infectious agents 17,18 . Monocytes from CL patients express more TLR2, TLR4 and TLR9 ex vivo or in soluble leishmania antigen (SLA)-stimulated cultures than cells from healthy subjects (HS) 19,20 . The elevated expression of TLR2 and TLR4 by intermediate monocytes from L. braziliensis-infected CL patients in comparison to HS has been associated with TNF production 21 . On the other hand, the neutralization of TLR4 was found to decrease TNF production by PBMCs infected with L. braziliensis 22 . While monocytes from CL patients produce more reactive oxygen species following exposure to L. braziliensis than HS monocytes 19 , less leishmania killing was observed in CL patients compared to subjects with asymptomatic L. braziliensis infection 23 . Parasite survival and proliferation in monocytes are enhanced by molecules such as superoxide dismutase (SOD) and prostaglandin 2 (PGE2), which are both enhanced in CL and visceral leishmaniasis; interestingly, the literature contains no reports on these molecules in DL 24,25,26 .
Parasite factors are also known to in uence the pathogenesis of leishmaniasis. As L. braziliensis is polymorphic, dissimilarity among strains has been associated with different clinical forms of disease, as well as failure to antimonial therapy 27,28,29 . Supporting the notion that differences among same-species leishmania may in uence immune response, SLA prepared using L. braziliensis isolates from DL patients was found to induce higher TNF production in mononuclear cells from both CL and DL patients than SLA similarly prepared using isolates of L. braziliensis from CL patients 30 . Moreover, L. braziliensis isolates obtained from DL patients were less internalized by neutrophils from HS, and induced lower oxidative burst and decreased expression of neutrophil activation markers 31 . Nonetheless, the literature contains scarce data on monocyte function in DL. The present study endeavored to compare the ability of DL and CL isolates to penetrate and survive in monocytes, as well as to induce molecules related to the pathogenesis of DL.

Results
The demographic and clinical features of the 24 DL and 24 CL patients included in the study are shown in Table 1. No differences were detected with regard to age, gender or size of the largest lesion. As expected, higher numbers of lesions were found in DL patients compared to CL, in addition to longer illness duration and a greater occurrence of mucosal disease.
Ability of DL and CL L. braziliensis Isolates to Infect Monocytes from DL and CL Patients Similar numbers of amastigotes are seen in biopsied ulcer samples from DL and CL patients 11 . However, multiple lesions in DL patients imply greater total numbers of parasites compared to CL. Here we infected monocytes obtained from DL and CL patients with isolates of DL or CL (Fig. 1). Regardless of donor cell origin, we identi ed higher frequencies of infected cells and greater number of amastigotes per 100 cells in monocytes infected with isolates from DL compared to CL at both 2 and 48 hours after infection (Fig. 1A). The median frequency of DL monocytes infected with the CL isolate was 46% (30-58 cells) at 2 hours versus 58% (45-76 cells) with the DL isolate (p < 0.01), compared to monocytes from CL patients: 45% (24-70 cells) for the CL isolate compared to 54% (37-84 cells) for the DL isolate (p < 0.001). Similar patterns were observed at 48 hours, yet a higher frequency of DL monocytes infected with the DL isolate observed after 48 hours of infection as compared to the earlier timepoint.
With respect to HS cells, the frequency of monocytes infected with the DL isolate was higher at 2 hours (p < 0.05) compared to the CL isolate ( Supplementary Fig. 1).
Regarding parasite load after 2 hours of infection, the number of parasites in DL monocytes infected with the CL isolate was 64 (range: 48-101) amastigotes/100 cells versus the DL isolate: 107 (range: 66-188) amastigotes/100 cells (p < 0.05). The number of amastigotes/100 cells was also higher in CL monocytes infected with the DL isolate compared to CL (P < 0.05) at this same timepoint. Again, higher numbers of intracellular parasites were seen in DL monocytes after 48 hours of infection compared to 2 hours.

Viability of extracellular promastigotes
Parasite viability was evaluated in the supernatants of cell cultures 48 hours after infection to determine whether the higher frequency of infected cells and elevated number of parasites detected in monocytes infected with DL isolates could lead to cell lysis and consequently the release of promastigotes in supernatants of monocyte cultures (Fig. 2). Higher numbers of parasites were quanti ed in the supernatants of DL and CL monocyte cultures (p < 0.05) when isolates obtained from DL patients were used for infection ( Figs However, in cells from DL this effect was higher. This indicates that upon L. braziliensis infection monocytes from DL had a lower ability to kill leishmania than CL monocytes.

Oxidative burst
To determine whether the induction of respiratory burst differed in parasites obtained from DL and CL patients, and to determine the impairment of this capability of DL monocytes, DL, CL and HS monocytes were infected with isolates from DL and CL patients. Median DHR expression, represented by mean uorescence intensity (MFI) values, in monocytes from DL patients infected with a CL isolate was 3715 (730-5310) versus 4790 (4000-8430) when infected with a DL isolate (P < 0.05). DHR expression in CL monocytes infected with a CL isolate was 2870 (330-4400) versus 4140 (3180-6700) with DL P < 0.05 (Fig. 3). No differences in DHR expression were seen between DL and CL monocytes infected with either type of isolate. In HS cells, infection with the DL isolate induced higher oxidative burst compared to CL (Supplementary Fig. 2) Expression of TLR2 and TLR4 in monocytes infected with DL and CL isolates We found no differences in the ex vivo expression of TLR2 and TLR4 when comparing monocytes from DL and CL patients (data not shown). Figure 4 illustrates TLR2 and TLR4 expression after infection with each L. braziliensis isolate. While monocytes from DL patients expressed elevated levels of TLR2 and TLR4 following infection with a DL isolate compared to CL: 1425 (274-2350) and 2384 (270-3542) versus 853 (91-1633) and 1544 (235-3028), respectively (P < 0.05), similar expression for both receptors was observed when DL or CL isolates were used to infect CL monocytes (P > 0.05). Additionally, TLR2 and TLR4 expression was higher in DL monocytes compared to CL monocytes after infection with a DL isolate (p < 0.01 and p < 0.05). In HS cells, infection with a DL isolate induced higher expression of TLR compared to CL ( Supplementary Fig. 3) Expression of Proin ammatory Cytokines Figure 5 shows the median MFI values obtained from DL and CL monocytes expressing TNF, CXCL9 and CXCL10 following infection with isolates of L. braziliensis from DL or CL patients.
When comparing the monocytes of patients with the different clinical forms, infected with the same L.braziliensis isolate, we did not observe any signi cant differences regarding the expression of CXCL9 and CXCL10.
We found no differences in IL-10 production regardless of monocyte origin (CL or DL) or the isolate used for infection (DL vs. CL) (data not shown).

Discussion
DL is an emergent and severe form of L. braziliensis infection 1,10,37 . While the mechanism underlying parasite dissemination has yet to be identi ed, it is clear that this does not occur at the time of infection.
Indeed, multiple acneiform and papular lesions suddenly appear only days or weeks after a primary ulcerated lesion; dissemination has been associated with fever and chills lasting 1-2 days. 10 As L. braziliensis is polymorphic, distinct genotypic characteristics among isolates have been linked to different clinical forms of disease, e.g. cutaneous, mucosal, DL or atypical CL 27,28,33 . Here we attempted to evaluate whether infection with DL isolates provokes different behavior compared to CL in terms of parasite internalization and multiplication, as well as monocyte activation and the production of in ammatory molecules. The present results indicate that L. braziliensis isolates from DL patients exhibit a greater ability to penetrate and multiply in monocytes compared to CL, in spite of higher respiratory burst induction and enhanced proin ammatory cytokine production. Additionally, host factors also play a role in the parasite dissemination, as monocytes from DL is more permissive to the infection and allow a greater parasite multiplication than CL cells upon infection with a DL isolate. Moreover, cells from DL expressed more TLR-2 and TLR-4 than cells from CL patients infected with a DL isolate, and produce higher levels of PGE2 than CL cells upon stimulation with SLA.
In the rst 48 hours after in vitro infection with L. braziliensis, the percentage of infected monocytes/macrophages and the number of intracellular parasites increases, reaches a plateau and then begins to decrease 19,38 . Parasite internalization is observed via the quanti cation of these parameters in the rst two hours of infection. After this time, the percentage of infected cells and numbers of parasites re ect the ability of leishmania to multiply inside host cells, as well the capacity of these infected cells to kill parasites. Here, we demonstrate that experimental infection using isolate from DL patient enables greater monocyte penetration and survival compared to isolates from CL, regardless of the origin of source cells, i.e. DL or CL monocytes, providing evidence of the capability of genotypic differences among L. braziliensis to interfere in parasite internalization and multiplication.
Parasite proliferation inside phagocytic cells leads to cell lysis and the release of leishmania 36 . Our results document the greater viability of DL isolate compared to CL in the supernatants of DL, CL and HS monocyte cultures. Interestingly, and perhaps more important than genotypic differences among parasites, monocytes from DL patients were observed to allow enhanced leishmania multiplication, as higher numbers of viable promastigotes were observed in the supernatants of cultured monocytes from DL patients, in comparison to CL, following infection with a DL isolate. This results provides evidence that DL monocytes were more permissive to leishmania survival, regardless of the source of L. braziliensis, i.e. isolates from DL or CL patients. SOD enhances parasite multiplication in macrophages and SOD is highly expressed in host tissue during CL infection by L. braziliensis 32 . In addition, in CL caused by L. amazonensis, SOD plasma levels constitute a predictor of failure to meglumine antimoniate therapy 24 . More recently, PGE-2 has been associated with leishmania proliferation and survival 25,26,39 . Here we found no differences in SOD serum levels but observed that, upon stimulation with SLA, monocytes from DL patients produced higher levels of PGE-2 than cells from CL patients. This nding lends support to the role of monocytes, since origin, i.e. DL vs. CL, as was shown to in uence parasite survival.
Monocytes from CL patients present higher oxidative burst and also produce more reactive oxygen species (ROS) and nitric oxide (NO) than cells from HS; still, these phenomena are not su cient to control parasite multiplication and may still lead to pathology 19 . The importance of NO in the killing of L. infantum and L. amazonensis has been documented, as well as that of ROS in the control of L. braziliensis proliferation 15,40,41 . Ávila et al. showed that L. braziliensis promastigotes and amastigotes isolated from CL and ML patients produced similar amounts of NO in culture. However, promastigotes from ML isolates were found to be more resistant to NO and H 2 O 2 than CL parasites 42 . Our results document that infection with DL isolates induces higher respiratory burst in monocytes from both DL and CL patients compared to isolates from CL, yet the observed enhancement in oxidative burst did not inhibit parasite multiplication, which suggests that DL isolates are less susceptible to monocyte killing than CL.
While TLRs are known to participate in host defense mechanisms, the expression of TLRs has also been associated with in ammatory and autoimmune diseases 43 . Our results show that monocytes from DL patients infected with DL isolates express more TLR2 and TLR4 compared to CL isolates. However, we did not similarly document this in CL monocytes. As cells from DL expressed more TLRs upon infection with DL isolates, but also exhibited less capability to kill leishmania, we suggest that the exaggerated in ammatory response observed in DL may likely be more closely related to pathology than protection.
Proin ammatory cytokines, such as TNF, CXCL9 and CXCL10, are mainly produced by monocytes/macrophages. In CL patients, these cells produce higher levels of cytokines than healthy subjects 38 . Elevated systemic production of CXCL9 has been documented in sera from DL patients compared to CL 10 . Additionally, upon stimulation in PBMCs of patients with both forms of disease, SLA obtained from isolates of DL patients induced higher TNF and IFN-γ expression than SLA from CL 30 . The present work expanded on these observations by demonstrating that DL isolate induce higher levels of TNF, CXCL9 and CXCL10 expression than CL isolate in cultured monocytes from both CL and DL patients. Indeed, it appears controversial that infection using DL isolate induces a pronounced proin ammatory response in monocytes, yet at the same time permits parasite survival inside phagocytic cells. However, it is important to consider that proin ammatory cytokine production by monocytes has not been de nitively linked to parasite killing in CL caused by L. braziliensis 38 .
Our data indicate that parasite dissemination in DL occurs due to parasite multiplication, cell death and the release of amastigotes that infect monocytes at different sites of the skin. However, we cannot rule out the possibility that parasite dissemination may also occur through the metastasis of infected monocytes from the original lesion site to other areas of the body. In cancer, in ammation in uences metastasis. More speci cally, the production of CXCL9 and CXCL10, among other proin ammatory cytokines, has been associated with the severity of melanoma and metastasis 44,45 . Here we expanded on a previous report that documented higher pro-in ammatory cytokine production induced by DL isolates compared to CL isolates 30 . In light of the lack of evidence that in ammation correlates with leishmania killing in L. braziliensis infection, it is important to consider the possibility that in ammation may hold in uence over parasite dissemination. DL, a severe and emergent form of L. braziliensis infection, has been associated with high rates of failure to antimony therapy. Our results show that parasite dissemination can be in uenced by host and parasite factors, and that parasite multiplication in macrophages is highly linked to parasite dissemination. As in ammation has been associated with the pathology of L. braziliensis infection, emphasis has been placed on a combined regimen of chemotherapy with immunomodulatory agents in the treatment of TL 46,47 . In addition to offering insight into the pathogenesis of DL, the present results point to the necessity of identifying novel therapeutic agents capable of enhancing leishmania killing by macrophages in order to enable the control leishmaniasis.

Patients
A total of 24 patients with DL and 24 with CL were included; all individuals sought medical attention at the Corte de Pedra Health Clinic, located in the municipality of Presidente Tancredo Neves (Bahia-Brazil), between September 2017 and May 2019. For comparison purposes, we included 12 healthy subjects (HS) without exposure to leishmania from a non-endemic area. DL patients presented more than 10 acneiform, popular, and ulcerated lesions, in two or more non-contiguous parts of the body (head, trunk, arms or legs). CL patients presented 1-3 ulcers with raised borders. Infection was diagnosed via detection of L.
braziliensis DNA by PCR in biopsied ulcer tissue samples 34 in conjunction with parasite isolation in culture or identi cation by histopathologic analysis. All experiments were performed prior to the administration of therapy.

Ethics Statement
The present research protocol received approval from the Institutional Review Board of the Federal University of Bahia, and was approved by the National Commission for Ethics in Research (CONEP) in Brazil (protocol number 2.114.874). All subjects agreed to voluntarily participate and provided a written term of informed consent. All methods were performed in accordance with the guidelines and regulations stipulated by CONEP.

Parasites
Species determination was based upon HSP70 PCR-restriction fragment length polymorphism and later con rmed by serial real-time quantitative PCR 34 . The genotyping of L. (V.) braziliensis recovered from CL and DL lesions was performed as previously described 27,33 . The isolates from CL and DL used to infect cells were genotyped according to the haplotypes of polymorphic nucleotides in the locus CHR28/455451, previously shown to distinguish L. braziliensis strains 27 . The CL and DL L. (V.) braziliensis isolates were cultured in biphasic Novy-MacNeal-Nicolle medium with liver infusion tryptose at 26 °C for 1-2 weeks. The suspension was then transferred to Schneider's medium containing 10% heat-inactivated fetal calf serum and 2 mM l-glutamine, and re-incubated at 26 °C for up to two weeks. Parasites were frozen in liquid nitrogen without any further subculturing in 10% dimethyl sulfoxide with 90% growth medium, and thawed prior to use in experimentation.

Isolation of Human Peripheral Blood Cells and Infection with L. braziliensis Isolates
Peripheral blood mononuclear cells (PBMC) were isolated from DL and CL patients and HS. Heparinized venous blood was layered 35 and resuspended in RPMI 1640 medium supplemented with 5% fetal calf serum and antibiotics (GIBCO BRL, Grand Island, NY USA). PBMCs (1 × 10 6 cells/tube) were infected with stationary-phase L. braziliensis isolates at a ratio of 5:1. After 2 hours of infection at 37 °C under 5% CO 2 , extracellular parasites were removed following centrifugation. Cells were placed in complete RPMI 1640 medium and incubated for an additional 48 hours. Finally, the numbers of infected cells and intracellular parasites were determined by microscopic evaluation of 100 monocytes following Romanowsky staining of cytocentrifuge preparations.

Evaluation of Parasite Viability
After 48 hours of infection with L. braziliensis isolates, PBMCs were washed and the medium was replaced with 0.5 ml of Schneider's medium (Sigma-Aldrich) supplemented with 10% fetal calf serum.
Cells were cultured at 26 °C for an additional 48 h. Viable parasites was determined by counting extracellular motile promastigotes using a hemocytometer 36 .
Determination of PGE2 and SOD PGE2 production was evaluated using reagents purchased from R&D Systems (Minneapolis, USA) in the supernatants of PBMCs (3 × 10 6 cells/ml) stimulated with SLA (5 µg/ml). Results are expressed in pg/ml. SOD serum levels were determined using a Human Cu/Zn Superoxide Dismutase ELISA kit (ABCAM. Cambridge Science Park, UK).

Monocyte Expression of TLR2 and TLR4
Monocyte surface expression of TLR2 and TLR4 was analyzed both ex vivo and in vitro after infection with L. braziliensis for 2 hours. The following antibodies were used: APC-conjugated anti-CD14 (APC clone M5E2, BD Pharmingen); PE-conjugated anti-TLR2 (clone TL2.1); PE-conjugated anti-TLR4 (clone HTA125) (eBioscience, San Diego, CA, USA). A total of 200,000 cells per tube were evaluated on a FACS Canto II ow cytometer (BD); data analysis was performed using FlowJo software (Tree Star Inc).
Evaluation of cytokine production PBMCs were either infected with different isolates of L. braziliensis or left uninfected. To perform ow cytometry, 10 6 PBMCs were stained with a uorochrome-conjugated CD14 surface marker antibody (APC clone M5E2, BD Pharmingen) and xed in 2% formaldehyde. For intracellular staining, xed cells were permeabilized using a cyto x/cytoperm kit (BD-Bioscience) and stained intracellularly with PE-conjugated anti-TNF (clone Mab11, eBioscience), PE-conjugated anti-CXCL9 (clone B8-11, BD Biosciences) and PEconjugated anti-CXCL10 (clone J034D6, BioLegend) antibodies. A total of 200,000 cells per tube were evaluated on a FACS Canto II ow cytometer (BD); data analysis was performed using FlowJo software (Tree Star Inc).

Statistical Analysis
Categorical variables with normal distribution were analyzed using the Student's T test. Comparisons between different isolates within a single group were performed using Wilcoxon's signed-rank test, while comparisons between groups were performed using the nonparametric Mann-Whitney U test. Differences among three or more groups were assessed by analysis of variance (Kruskal-Wallis) with Dunn´s posttest. Statistical signi cance was considered when p < 0.05. Data analyses were performed using GraphPad Prism 5.0 (GraphPad Software, Inc., San Diego, CA, USA).