Protective immunity to cutaneous leishmaniasis is mediated by IFN-γ–secreting CD4+ Th1 cells. IFN-γ binds to its receptor on Leishmania-infected macrophages, resulting in their activation, production of NO, and subsequent destruction of parasites. This study investigated the role of Semaphorin 3E (Sema3E) in host immunity to Leishmania major infection in mice. We observed a significant increase in Sema3E expression at the infection site at different timepoints following L. major infection. Sema3E-deficient (Sema3E knockout [KO]) mice were highly resistant to L. major infection, as evidenced by significantly (p < 0.05–0.01) reduced lesion sizes and lower parasite burdens at different times postinfection when compared with their infected wild-type counterpart mice. The enhanced resistance of Sema3E KO mice was associated with significantly (p < 0.05) increased IFN-γ production by CD4+ T cells. CD11c+ cells from Sema3E KO mice displayed increased expression of costimulatory molecules and IL-12p40 production following L. major infection and were more efficient at inducing the differentiation of Leishmania-specific CD4+ T cells to Th1 cells than their wild-type counterpart cells. Furthermore, purified CD4+ T cells from Sema3E KO mice showed increased propensity to differentiate into Th1 cells in vitro, and this was significantly inhibited by the addition of recombinant Sema3E in vitro. These findings collectively show that Sema3E is a negative regulator of protective CD4+ Th1 immunity in mice infected with L. major and suggest that its neutralization may be a potential therapeutic option for treating individuals suffering from cutaneous leishmaniasis.
The subset of CD4+ T cell response mounted by a host following infection with Leishmania major largely influences the outcome of the infection (1). Resolution of cutaneous lesions and acquired resistance to reinfection following primary L. major infection is directly correlated with the frequency of effector CD4+ Th1 cells and quantity of IFN-γ secreted at both the infection site and its draining lymph nodes (2). IFN-γ secreted by CD4+ Th1 cells is needed for activating macrophages to produce NO, which mediates effective killing of the intracellular parasites (3). In contrast, lesion progression and heavy parasite burden have been associated with CD4+ Th2 cells and regulatory T cell (Treg) responses, which are characterized by the secretion of IL-4 (4) and IL-10 (5), respectively. These cytokines inhibit classical macrophage activation, suppress NO production, and enhance the synthesis of polyamines, thereby creating an environment that favors parasite proliferation (6). The ability of CD4+ T cells to differentiate into IFN-γ–producing Th1 cells in the infected host is also strongly dependent on the quantity of IL-12 produced by the dendritic cells (DCs) (7). In addition, the upregulation of the transcription factor, T-bet, in naive CD4+ T cells during T cell activation is critical for their differentiation into effector CD4+ Th1 cells (8).
During L. major infection, both parasite and host factors interact to determine the disease outcome. Semaphorins are host proteins that are made up of ∼500 aa (9). They are phylogenetically classified into eight classes (9) and were previously thought to be involved only in axon guidance in the nervous system during embryogenesis (10). However, recent studies have shown that they play important physiologic and immunologic roles in the respiratory (11), gastrointestinal (12), and cardiovascular (13) systems. Recent studies have implicated Semaphorin 3E (Sema3E), one of the classes of semaphorins expressed in vertebrates (9, 14), as a major player in cell-mediated immunity. Deficiency of Sema3E resulted in enhanced DC migration to the lungs, leading to exacerbation of airway inflammatory responses in allergic asthma (15). Furthermore, DCs from plexin D1– (Sema3E receptor) deficient mice secreted higher levels of IL-12/IL-23p40 after LPS stimulation (16). In addition, in vitro–differentiated Th2 cells expressed significantly higher levels of Sema3E than Th1 cells (16). Collectively, these observations suggest that Sema3E influences CD4+ T cell differentiation and could favor the development of Th2 cells while suppressing Th1 immune responses.
In this article, we report that L. major infection significantly upregulates Sema3E secretion at the infection site. Genetic ablation of Sema3E leads to enhanced resistance to L. major infection, and this was associated with enhanced Th1 immune responses in their draining lymph nodes. Furthermore, we showed that the enhanced Th1 response was related to increased production and expression of IL-12 and costimulatory molecules by Sema3E-deficient CD11C+ cells. Sema3E deficiency significantly enhanced in vitro differentiation of naive CD4+ T cells into IFN-γ–secreting Th1 cells, which was reversed following the addition of recombinant Sema3E to the in vitro differentiation conditions. Collectively our results show that Sema3E is a host factor that contributes to susceptibility to L. major infection by suppressing the development of Th1 immune response.
Materials and Methods
The experiments conducted in this study were approved by the University of Manitoba Animal Care Committee and were carried out according to the regulation and guidelines of the Canadian Council on Animal Care (Protocol Number 17-007).
Six- to eight-week-old female C57BL/B6 mice were obtained from the Genetic Modeling Centre at the University of Manitoba. Sema3E knockout (KO) mice on C57BL/6 background were developed inhouse by crossing homozygous female and male Sema3E KO mice, which were originally obtained from Dr. C. Gu (Harvard Medical School, Boston, MA) (17).
Parasites and infection
L. major Fredlin (MHOM/80/Fredlin) strains were used for the entire study. Promastigotes were cultured at 26°C in M199 medium (Hyclone, Logan, UT) supplemented with 20% heat-inactivated FBS (Cansera, Mississauga, ON, Canada) and 100 U/ml penicillin/streptomycin. Stationary phase promastigotes from day 7 cultures were washed three times with sterile PBS, and 2 × 106 parasites were injected s.c. into the left hind footpad or left ear. The uninfected footpads and ears were injected with sterile PBS.
Measurement of lesion sizes and quantification of parasites
The lesion size was measured weekly with digital calipers. At designated time points, infected mice were sacrificed and parasite burden in the infected feet was quantified by limiting dilution assay as described previously (18).
Generation of bone marrow–derived CD11c+ cells and macrophages
Bone marrow–derived CD11c+ cells (BMDCs) were generated from bone marrow cells, as described by Lutz et al. (19). Briefly, bone marrow cells (2 × 105/ml) were plated in Petri dishes in complete medium (RPMI 1640 medium supplemented with 10% heat-inactivated FBS and 100 U/ml penicillin/streptomycin, 5 × 10−5 2-ME, and 2 mM l-glutamine) in the presence of 20 ng/ml GM-CSF. After 8 d, immature BMDCs were harvested from the Petri dishes, and flow cytometry was used to determine the percentage of CD11c+ cells (∼95%). To obtain macrophages, bone marrow cells (obtained as above) were cultured in 100 × 15–mm Petri dishes (BD Falcon) at 4 × 105 cells/ml in 10 ml of complete RPMI media supplemented with 30% L929 cell culture supernatant at 37°C. After 8 d, flow cytometry was used to determine the percentage of F4/80+ cells (∼90%).
Infection and stimulation of BMDCs and bone marrow–derived macrophages with L. major and TLR agonists
Bone marrow–derived macrophages (BMDMs) and BMDCs were infected in vitro with purified stationary phase L. major promastigotes in polypropylene tubes at a parasite to cell ratio of 5:1. In some studies, BMDCs were further stimulated in vitro with LPS (100 ng/ml; Sigma-Aldrich) or CpG (1 μg/ml; Sigma-Aldrich), and BMDMs were stimulated with rIFN-γ (10 ng/ml). The infected and/or stimulated cells were cultured at 37°C for various time points (ranging from 6 to 72 h) and assessed for parasite infectivity and costimulatory molecule expression by light microscopy and flow cytometry, respectively.
CD4+ T cell and BMDC cocultures
CD4+ T cells were purified (by negative selection) from pooled spleen and lymph node cell suspensions from PEPCK-specific TCR transgenic (Tg) mice (20) according to the manufacturer’s suggested protocol (StemCell Technologies). The purified CD4+ T cells (>92% purity) were stained with CFSE and cocultured with PEPCK peptide- (PEPCK335–351, NDAFGVMPPVARLTPEQ, 5 μM) pulsed BMDC obtained from wild-type (WT) and Sema3E KO mice (DC/T cell ratio of 1:100). After 4 d, proliferation (CFSE dilution) and IFN-γ secretion by CD4+ T cells were analyzed by flow cytometry.
Intracellular cytokine detection and Ag recall response
At different time points postinfection, mice were sacrificed, and single-cell suspensions were obtained from the spleens and draining lymph nodes as previously described (21). Cells were stimulated with PMA, ionomycin, and brefeldin A for 4 h and then stained with fluorochrome-conjugated Abs against CD3 and CD4 molecules. Thereafter, the cells were fixed, permeabilized with saponin, and stained intracellularly for IFN-γ and IL-4 using appropriate fluorochrome-conjugated Abs. For the in vitro Ag recall response, the single-cell suspensions of the spleens and draining lymph nodes were stimulated with soluble Leishmania Ag (SLA; 50 μg/ml) for 3 d at 37°C, and the supernatant fluids were collected and stored at −20°C until analyzed for cytokines by ELISA.
The concentrations of IFN-γ, IL-4, IL-10, and IL-12p40 in the cell culture supernatant fluids were measured by sandwich ELISA using paired Abs from BD Pharmingen (San Jose, CA) according to manufacturer’s protocols. The sensitivities of the ELISAs were 5 pg/ml for IL-4 and IL-10 and 30 pg/ml for both IL-12p40 and IFN-γ.
Real-time PCR assay
At the designated time points (day 1, 2, and 7 postinfection), the ears of infected or PBS-treated mice were harvested and evaluated for Sema3E expression by PCR. The ear samples were further homogenized, and the resultant RNA was reverse transcribed to cDNA using the reverse transcription (RT) kit (Life Technologies–BRL, Gaithersburg, MD). In detail, ear samples were kept in 1.5-ml Eppendorf tubes containing 1 ml of TRIzol (Thermo Fisher Scientific) reagent on ice and properly mixed and kept for 5 min at room temperature. Two hundred microliters of chloroform was added into the Eppendorf tubes containing a mixture of TRIzol and digested ear tissue samples. Tubes were vigorously mixed for 15 s, kept in room temperature for ∼3 min, and subsequently centrifuged for 15 min at 12,000 × g. The resultant aqueous phase of the solution was poured into sterile RNase-free tubes, after which 500 μl of isopropyl alcohol was added. The tubes were incubated for 10 min at room temperature and then spun for 10 min at 11,000 × g. The supernatants were discarded and replaced with 75% alcohol. The resultant RNA pellets were dissolved in RNase-free water and stored at −20°C. To reverse transcribe RNA into cDNA, 10 μl of total RNA was added into designated wells in a 96-well plate already containing 10 μl of reverse transcription master mix (25× dNTP + nuclease-free water + 10× RT random primers + RNase inhibitor + Multiscribe Reverse Transcriptase + 10× RT buffer). The thermal cycler was programmed according to the manufacturer’s protocols, and the reverse transcription was done. The primers used were 5′-AAAGCATCCCCAACAAACTG-3′ (forward) and 5′-GTCCAGCAAACAATTCACTACCA-3′ (reverse) (Invitrogen).
CD4+ T cell purification and in vitro Th1, Th2, and Treg polarization
Pooled single-cell suspensions from the spleens and lymph nodes of WT and Sema3E KO mice were labeled with CFSE, as previously described (20). Thereafter, CD4+ T cells were isolated by negative selection using a stem cell CD4+ T cell isolation kit according to the manufacturer’s protocol. The purity of isolated CD4+ cells was ≥98%. Highly enriched CFSE-labeled CD4+ T cells were resuspended at 2 × 106 cells/ml in complete medium (DMEM medium supplemented with 10% heat-inactivated FBS and 100 U/ml penicillin/streptomycin, 5 × 10−5 2-ME, and 2 mM l-glutamine). One hundred microliters of the cell suspension (2 × 105 cells) was cultured under the following polarization conditions: Th0 (1 μg/ml anti-CD3, 1 μg/ml anti-CD28), Th1 (1 μg/ml anti-CD3, 1 μg/ml anti-CD28, 20 ng/ml rIL-12, 10 μg/ml anti–IL-4), Th2 (1 μg/ml anti-CD3, 1 μg/ml anti-CD28, 10 ng/ml rIL-4, and 10 μg/ml anti–IL-12), and Treg (1 μg/ml anti-CD3, 1 μg/ml anti-CD28, 10 ng/ml rTGF-β, 10 μg/ml anti–IFN-γ, 10 μg/ml anti–IL-12, and 10 μg/ml anti–IL-4). In some studies, recombinant Sema3E (100 ng/ml) was added to the polarization conditions. After 4 d, the cells were assessed for IFN-γ secretion and Foxp3 expression by flow cytometry. In some studies, the cells were centrifuged, and the levels of IFN-γ, ΙL-4, and IL-10 in the culture supernatant fluids were assessed by ELISA. The cells were lysed with TRIzol, and total RNA was extracted and used to quantify the mRNA levels of IFN-γ, IL-4, IL-10, Foxp3, GATA-3, and T-bet by quantitative PCR (qPCR).
Bone marrow chimera study
Single-cell suspensions of WT and Sema3E KO bone marrow cells were resuspended to 10 × 106 cells per 150 μl and injected i.v. into lethally irradiated (900 rad for 2 min) recipient WT and Sema3E KO mice 4 h prior to cell transfer. The blood of the recipient mice was monitored (by flow cytometry) weekly to check for full reconstitution with donor cells (∼8–10 wk). Thereafter, the mice were infected with L. major promastigotes, and progression of lesion development was monitored.
C57BL/6 mice were injected intradermally in the ears with either PBS (uninfected control) or 2 × 106 L. major parasites resuspended in 10 μl of PBS. Mice were sacrificed after 1, 2, or 7 d, and ears were harvested. The tissues were fixed for 1 h at 4°C in 4% (vol/vol) paraformaldehyde in PBS and incubated at 4°C for 1 h in 10 and 20% (wt/vol) sucrose in PBS and then in 30% sucrose overnight. Tissues were embedded in OCT compound (Thermo Fisher Scientific) and cut into 10-μm sections using a cryostat and mounted onto microscope slides. The slides were washed and blocked with Fc blocker (Innovex), 4% v/v mouse serum (ImmunoReagents), 4% goat serum, and 4% donkey serum. The primary Ab used was goat anti-mouse Sema3E (R&D Systems, Oakville, ON, Canada) at 1:75 dilution. Secondary Abs used were AF568-conjugated donkey anti-goat (Abcam, Toronto, ON, Canada) at 1:400 dilution and AF488-conjugated goat anti-rat (Abcam) at 1:400 dilution. Slides were stained with Hoechst 33342 (Molecular Probes, Eugene, OR) for 30 min at 1:2000 dilution and mounted with ProLong Gold (Invitrogen, Waltham, Massachusetts). Images were acquired using the Zeiss Axio Observer confocal microscope. Fluorescence intensity was determined with ImageJ software (National Institutes of Health, Bethesda, Maryland).
The data shown in this study were reported as the mean ± SEM. The differences between the lesion sizes, parasite burdens, quantity of secreted cytokines, and frequency of various cells between the different groups of mice were determined by Student t test and one-way ANOVA. When p ≤ 0.05, the result is considered to be significant.
L. major infection modulates the expression of Sema3E at the infected cutaneous site
DCs, macrophages, and CD4+ T cells, which are important immune cells implicated in Leishmania immunity (7, 22–24), have been shown to express Sema3E (16). These cells are rapidly recruited to the cutaneous sites of infection following L. major infection (22, 23). We, therefore, sought to investigate whether L. major infection regulates the expression of Sema3E and, if it does, determine the cells that express Sema3E at the infected cutaneous site. We injected L. major intradermally into the ears of WT mice and assessed Sema3E mRNA level by real-time PCR. We observed a significant increase in Sema3E mRNA at days 1 and 2 postinfection compared with the control (PBS-injected) groups (Fig. 1A). Interestingly, by day 7 postinfection, the expression of Sema3E was significantly downregulated compared with the PBS-treated group (uninfected mice) (Fig. 1A). We confirmed the expression of Sema3E in both infected ear tissue and F4/80+ cells by confocal microscopy (Fig. 1B). Using Image J software, we assessed the intensity of Sema3E protein expression and observed a similar trend as in mRNA expression at the designated time points (Fig. 1C). These results suggest that infection with L. major induces Sema3E expression in host cells early during infection, but this is downregulated by 7 d postinfection, possibly in an attempt to create a more anti-Leishmania environment.
Deficiency of Sema3E results in enhanced host resistance to L. major infection and alters the quality of immune cell infiltration at the site of infection
Some key immune cells, such as DCs, macrophages, and CD4+ T cells that play critical roles in immunity to L. major infection (7, 22–24), also express Sema3E (16). Because L. major infection induced early expression of Sema3E that was later downregulated as the disease progressed (Fig. 1), we investigated the impact of Sema3E in the pathogenesis of L. major infection. We infected WT and Sema3E KO mice with L. major and monitored disease (lesion) progression for several weeks postinfection. Sema3E KO mice showed a significant (p < 0.001) decrease in their lesion size throughout the infection period compared with their WT counterparts (Fig. 2A). Consistent with this, infected Sema3E KO mice had significantly (p < 0.001) lower parasite burden at 5 and 8 wk postinfection compared with their infected WT counterparts (Fig. 2B).
The composition of cells infiltrating the site of L. major infection greatly influences the disease outcome. Whereas the presence of Th1 cells results in decreased parasite burden, the influx of inflammatory cells (such as monocytes and neutrophils) has been reported to contribute to the exacerbation of cutaneous lesion. Therefore, we investigated whether Sema3E regulates infiltration of immune cells into the cutaneous site early (10 d) during L. major infection and the type of cells involved. We observed that infected WT mice contained significantly (p < 0.05) more neutrophils and monocytes in their infected ears compared with Sema3E KO mice (Fig. 2C, 2D). In contrast, infected ear tissues of Sema3E KO mice contained more CD4+ T cells and MHC class II+CD11c+ cells than their infected WT counterpart mice (Fig. 2C, 2D). Collectively, these results suggest that deficiency of Sema3E enhances resistance to L. major infection and alters the quality of inflammatory cells that infiltrate into the site of infection.
Enhanced resistance of Sema3E KO mice to L. major infection is mediated by immune cells
Sema3E is expressed by both immune and nonimmune (structural) cells, including epithelial and retinal ganglion cells (9). Therefore, we wished to determine whether immune cells were responsible for the enhanced resistance to L. major infection in Sema3E KO mice. Bone marrow cells from WT and Sema3E KO mice were adoptively transferred to lethally irradiated WT and Sema3E KO mice, and following full reconstitution of the immune repertoire (8 wk), the recipient mice were infected with L. major parasites. WT and Sema3E KO mice that received bone marrow cells from Sema3E KO mice both displayed enhanced resistance (significantly smaller lesion size) similar to Sema3E KO mice. In contrast, Sema3E KO mice that received bone marrow cells from WT mice lost their enhanced resistance and displayed a phenotype similar to WT mice (Fig. 3A). The reduction in lesion size observed in Sema3E KO and WT mice that received Sema3E KO bone marrow cells was associated with significantly (p < 0.001) decreased parasite burden compared with WT mice or Sema3E KO mice that received WT bone marrow cells (Fig. 3B). Because the lethally irradiated Sema3E KO mice that received WT bone marrow cells had Sema3E KO stromal cells but could not control infection, this result indicates that the enhanced resistance observed in L. major–infected Sema3E KO mice is due to deficiency of Sema3E in immune cells.
Sema3E deficiency results in increased frequency of IFN-γ–producing CD4+ T cells following L. major infection
Th1 immune response manifesting as IFN-γ–producing CD4+ T cells is critical for clearance of L. major parasite and for host resistance to leishmaniasis. Thus, we sought to determine whether the enhanced resistance to L. major infection was related to superior Th1 CD4+ T cell response. At all times tested, the frequency of CD4+IFN-γ+ T cells (Th1) in the lymph nodes draining the infection sites and the quantity of secreted IFN-γ by cells from infected Sema3E KO mice were significantly higher compared with their infected WT counterparts (Fig. 4). Interestingly, infected Sema3E KO mice had significantly reduced numbers of CD4+IL-4+ (Th2) cells in their draining lymph nodes, and these cells secret significantly lower amounts of IL-4 following in vitro restimulation with SLA (Supplemental Fig. 1A–C). Collectively, these findings indicate that deficiency of Sema3E promotes anti-Leishmania Th1 immune response, which mediates effective parasite clearance, strongly suggesting that Sema3E may be a negative regulator of CD4+ Th1 response.
Sema3E-deficient macrophages are more responsive to IFN-γ in vitro
Macrophages are the major cells parasitized by Leishmania parasites in infected hosts (23–25), and they play a critical role in determining disease outcome. Therefore, we sought to investigate whether deficiency of Sema3E in macrophages affected infectivity and ability of L. major amastigotes to proliferate inside infected cells. There was no significant difference in both the number of parasites/infected cells and percentage of infection (number of infected macrophages per 100 cells) between Sema3E KO and WT macrophages (Fig. 5A, 5B). Following activation, macrophages secrete NO to eliminate intracellular parasites (25). We observed no significant difference in the quantity of NO secreted by L. major–infected WT and Sema3E KO macrophages (Fig. 5C). These results suggest that the enhanced resistance to L. major infection in Sema3E KO mice is not due to an enhanced intrinsic leishmanicidal capacity of their macrophages.
Next, we assessed whether L. major–infected WT and Sema3E KO macrophages responded differently to IFN-γ stimulation in vitro. L. major–infected WT and Sema3E KO macrophages were treated with rIFN-γ, and parasite numbers and NO production were assessed at different time points. We observed a significant decrease in parasite numbers and a corresponding increase in NO production by IFN-γ–treated L. major–infected Sema3E KO macrophages compared with their WT counterparts (Supplemental Fig. 2A–C). Collectively, these observations suggest that Sema3E KO macrophages are more responsive to IFN-γ–mediated activation and NO production, leading to a more-efficient parasite control.
Deficiency of Sema3E leads to increased expression of costimulatory molecules, IL-12p40 secretion, and T cell activation capacity by CD11c+ cells
CD11c+ DCs are one of the major players in the protective immunity to L. major infection (2, 7, 22, 26) because they are critical for priming and differentiation of naive CD4+ T cells into protective Th1 cells (27, 28). Therefore, we investigated the impact of Sema3E deficiency on CD11c+ cell activation (expression of costimulatory molecules and IL-12p40 production) following L. major infection. We observed a significant increase in the expression of CD80 and CD86 molecules and secretion of IL-12p40 by Sema3E KO BMDC compared with their WT counterparts following L. major infection (Fig. 6A–E) or stimulation with LPS (Supplemental Fig. 3A–F) or CpG (Supplemental Fig. 4A–F).
Next, we investigated whether the increased expression of costimulatory molecules and IL-12 production by CD11C+ cells could contribute to increased Th1 response following L. major infection. We cocultured PEPCK peptide- (Leishmania peptide) pulsed WT or Sema3E KO CD11c+ cells with CFSE-labeled naive CD4+ T cells from PEPCK-specific TCR Tg mice (20), and after 4 d, we assessed proliferation and IFN-γ production by flow cytometry and ELISA. There was a significant increase in the frequency of CD4+IFN-γ+ T cells (Fig. 6F, 6G) as well as quantity of IFN-γ secreted by T cells (Fig. 6H) after coculture with Sema3E KO CD11c+ cells compared with those cultured with WT CD11c+ cells. Taken together, these results show that Sema3E deficiency enhances the ability of CD11c+ cells to activate and differentiate Leishmania-specific CD4+ into IFN-γ–producing Th1 cells.
Sema3E directly regulates CD4+ Th1 differentiation in vitro
Resistance to L. major infection is directly associated with a strong Th1 response because IFN-γ (the signature Th1 cytokine) is the major effector cytokine that mediates parasite killing (3). We showed that the enhanced resistance of Sema3E KO mice to L. major infection was associated with increased frequency of IFN-γ–producing CD4+ (Th1) cells in their draining lymph nodes (Fig. 5). Although we showed that CD11c+ cells may play a role in this (Fig. 6F–H), and given that T cells express Sema3E and its receptor plexin D1 (16), it is plausible that direct Sema3E signaling in naive CD4+ T cells may block their intrinsic propensity to differentiate into Th1 cells. To investigate this, we differentiated naive WT and Sema3E KO CD4+ T cells into Th1 cells in vitro in the presence or absence of Sema3E. We observed a significant increase in the frequency of CD4+IFN-γ+ (Th1) cells, T-bet expression, and amount of IFN-γ secreted in the culture supernatant by Sema3E KO CD4+ T cells compared with their WT counterparts (Fig. 7). The addition of recombinant Sema3E to the cultures significantly suppressed the increased frequency of CD4+IFN-γ+ (Th1) cells, production of IFN-γ, and T-bet expression by Sema3E KO CD4+ T cells during in vitro Th1 differentiation (Fig. 7). Interestingly, cells from Sema3E KO mice produced lower amounts of IL-4 and expressed significantly lower GATA3 mRNA following in vitro Th2 differentiation (Supplemental Fig. 1D, 1E).
Altogether, these results strongly suggest that Sema3E is a negative regulator of CD4+ Th1 differentiation and cell-mediated immunity, and its deficiency enhances Th1 response leading to increased resistance in L. major infection.
Sema3E regulates Treg numbers and IL-10 production in vitro and in vivo
The suppression of host immune response during L. major infection has been shown to be mediated by IL-10–producing Tregs (5, 6). Given that Sema3E KO mice displayed enhanced Th1 immune response during L. major infection (Fig. 4), we sought to assess the effect of Sema3E deficiency on Tregs and IL-10 secretion during L. major infection. Draining lymph node cells from L. major–infected Sema3E KO mice had significantly lower numbers of CD25+Foxp3+ Tregs and produced lower IL-10 compared with their WT counterparts (Fig. 8A–C). Furthermore, naive CD4+ T cells from Sema3E KO mice display significantly decreased propensity to differentiate into IL-10–producing Tregs compared with their WT counterpart mice (Fig. 8D–F). This was associated with a significant decrease in mRNA levels of Foxp3 and IL-10 in Sema3E KO Tregs compared with their WT counterparts (Fig. 8G, 8H).
Resistance to cutaneous leishmaniasis is mediated by IFN-γ–producing CD4+ Th1 cells that are critical for activation of infected macrophages to produce NO responsible for destruction of intracellular parasites. In the current study, we assessed the contribution of Sema3E in host resistance to experimental cutaneous leishmaniasis caused by L. major infection. We found that compared with WT mice, Sema3E KO mice displayed enhanced resistance to L. major infection, as evidenced by significantly reduced lesion sizes and lower parasite burden at different times postinfection. The enhanced resistance of Sema3E KO mice was associated with reduced infiltration of inflammatory cells, decreased frequency of Tregs, and IL-4–producing CD4+ T cells as well as increased frequency of IFN-γ–producing CD4+ T cells, compared with those from WT mice. In vitro differentiation studies confirmed that CD4+ T cells from Sema3E KO have an intrinsically higher propensity to differentiate into Th1 cells, suggesting that Sema3E is a negative regulator of Th1 immune response and protective immunity against cutaneous leishmaniasis.
Although the semaphorins were previously thought to be primarily involved in axon guidance in the nervous system during embryogenesis (10), recent reports indicate that they also play essential roles in the optimal functioning of different physiologic systems, including respiratory (11), gastrointestinal (12), and cardiovascular (13) systems. In addition, several immune cells, including DCs and CD4+ (particularly Th2) cells, have been reported to express high levels of Sema3E (16). The observation that CD4+ Th2 cells [that are associated with host susceptibility to L. major infection (29)] express the highest levels of Sema3E compared with any other immune cells suggested that this molecule may play a crucial role in the pathogenesis of cutaneous leishmaniasis. Indeed, we observed a reduced Th2 immune response in L. major–infected Sema3E KO mice, which may have contributed to the enhanced Th1 immune response in these mice. Sema3E expression significantly increased very early following L. major infection in WT mice, and this was followed by rapid downregulation by day 7 postinfection, suggesting a tight regulation that may be necessary for host defense.
In addition to myeloid-derived cells, structural cells (including epithelial and retinal ganglion cells) also express Sema3E (9). Indeed, the expression of Sema3E by epithelial cells of the respiratory tract has been shown to be a major contributor to airway hyperresponsiveness and tissue remodeling during allergic inflammation and asthma (15). The infiltration of inflammatory cells to the site of infection contributes to lesion development and cutaneous tissue damage in L. major–infected mice (30). Sema3E have been shown to regulate cell migration (31), which is a critical process that controls infiltration of cells to inflammatory foci. We observed decreased numbers of inflammatory cells (neutrophils and monocytes) in the infected site of Sema3E KO mice, suggesting that Sema3E promotes cellular infiltration and inflammatory responses at the cutaneous site of infection. This observation differs from those made by Movassagh et al. (32) who showed that Sema3E is a chemorepellent and inhibits neutrophil migration in an experimental allergic asthma mouse model. This discrepancy may be related to differences in the experimental models: intracellular parasitic infection (Leishmania) versus house dust mites in allergic asthma.
Our bone marrow chimera studies clearly show that the impact of Sema3E deficiency on the pathogenesis of L. major infection was mediated by bone marrow–derived (immune) cells. We showed that deficiency of Sema3E significantly enhanced in vitro differentiation of CD4+ T cells into Th1 cells, strongly suggesting that Sema3E has a direct effect on T cell differentiation in WT mice. In line with this, we found that under Th1 polarizing conditions, the expression of T-bet, a key Th1 transcription factor, is significantly higher in CD4+ T cells from Sema3E KO mice compared with their WT counterparts. Furthermore, our data suggest that the enhanced resistance to L. major infection in Sema3E KO mice is not due to enhanced intrinsic ability of their macrophages to produce NO and kill parasites. Instead, we showed that Sema3E KO macrophages responded better to rIFN-γ, leading to more-effective parasite control. This increased responsiveness of Sema3E KO macrophages to IFN-γ could contribute to the enhanced resistance manifested as reduced parasites were observed in vivo in Sema3E KO mice during L. major infection.
DCs play a critical role in protective immune response to cutaneous leishmaniasis (22). They phagocytose Leishmania parasites, process and present their antigenic peptides to naive CD4+ T cells, and through the expression of costimulatory molecules (CD80, CD86) and IL-12 production, initiate the development of protective CD4+ Th1 response (3). We observed that Sema3E KO CD11c+ cells expressed significantly higher levels of costimulatory molecules, secreted more IL-12p40, and mediated more Ag-specific differentiation of Th1 cells than their WT counterparts. Collectively, these findings suggest that Sema3E signaling (as seen in Sema3E-competent CD11C+ cells) downregulates costimulatory molecule expression and proinflammatory cytokine production in CD11C+ cells, thereby suppressing their ability to activate and differentiate naive CD4+ T cells into CD4+ Th1 cells. In line with this, Holl et al. (16) reported that deficiency of Plexin D1 (which is the major receptor for Sema3E) on DCs resulted in enhanced IL-12 production. Thus, the enhanced resistance to L. major infection observed in Sema3E KO mice could in part be related to the increased capacity of their CD11C+ cells to induce differentiation of naive CD4+ T cell into protective CD4+ Th1 cells. Indeed, we found that deficiency of Sema3E enhances the ability of CD11C+ cells to activate Leishmania-specific CD4+ T cells, leading to their differentiation into IFN-γ–producing Th1 cells.
IFN-γ is a major effector cytokine secreted by CD4+ Th1 cells and is critical for resistance against L. major infection (4). Although we showed that enhanced DC function in the absence of Sema3E signaling contributed in part to the resistance, we also observed a significantly increased Th1 differentiation (T-bet expression, CD4+ IFN-γ+ T cells, and IFN-γ production) in highly purified CD4+ T cells from Sema3E KO mice compared with their WT counterparts following in vitro Th1 differentiation. This increased T-bet expression and CD4+ IFN-γ+ T cells, and IFN-γ production was directly suppressed following the addition of recombinant Sema3E into the cultures, demonstrating that Sema3E directly acts on CD4+ T cells to downregulate Th1 differentiation.
Although in vitro, we found that Sema3E directly affected CD4+ Th1 differentiation negatively; we also observed that Sema3E KO CD11c+ cells expressed higher costimulatory molecules and produced more IL-12p40, leading to an increased capacity to drive differentiation of naive Leishmania-specific CD4+ T cells into IFN-γ–producing Th1 cells. Thus, it is conceivable that this increased numbers of CD11c+ cells and elevated CD4+ T cell responses in the absence of Sem3E collectively contributed to the enhanced resistance of Sema3E KO mice to L. major infection. The enhanced CD11c+ cell response could directly enhance T-bet expression and IFN-γ production by CD4+ T cells. In line with this, it was recently shown that exposure of DCs to Mycobacterium Ag PPE39 led to increased ability to enhance T-bet and IFN-γ expression in naive CD4+ T cells (33). It is conceivable that the production of Sema3E following L. major infection in WT mice and its signaling after binding to its canonical receptor (Plexin D1) on CD4+ T cells suppress T-bet expression, resulting in downregulation of IFN-γ production and impaired Th1 response. In the absence of Sema3E signaling (as in Sema3E KO mice), unhindered T-bet expression results in increased differentiation of CD4+ Th1 cells and IFN-γ production, leading to increased leishmanicidal activity and enhanced resistance to L. major infection.
IL-10–producing Tregs have been shown to contribute to immunosuppression during L. major infection by suppressing Th1 immune response (5, 6). We observed a significant reduction in the frequency of Tregs in the spleens and draining lymph nodes of both naive (uninfected) and L. major–infected Sema3E KO mice, and this was associated with decreased secretion and mRNA expression of IL-10. Because previous studies have shown that increased Treg numbers is associated with decreased Th1 response during L. major infection, it is conceivable that one of the mechanisms by which Sema3E promotes disease pathogenesis may be by increasing Treg numbers, which in turn suppresses effective Th1 immune response. Thus, in the absence of Sema3E as present in Sem3E KO mice, the reduced numbers of Tregs and concomitant decrease in IL-10 result in more-effective IFN-γ responsiveness, leading to enhanced resistance to L. major infection. Further studies are warranted to specifically and definitively ascertain whether the reduction of Tregs in the absence of Sem3E critically contributes to the enhanced resistance of these mice to L. major infection.
Recent reports have shown that Sem3E negatively regulates Th17 and IL-17 response in vitro and in vivo. Sema3E levels were significantly reduced in colonic tissues from ulcerative colitis patients and colonic mice and negatively correlated with proinflammatory cytokine and IL-17 levels (12). Sema3E KO mice had exacerbated colonic inflammation that was associated with increased production of IFN-γ and IL-17 by draining lymph node cells (12). Similarly, Sema3E KO mice produced higher levels of IL-17 following HDM challenge (15). Although the role of IL-17 in the pathogenesis of cutaneous leishmaniasis is controversial (34–36), a preponderance of evidence suggests that it may play a protective role in Leishmania-infected mice (37–40). Indeed, we have found that deficiency of PTX3 results in enhanced IL-17 response, which synergizes with IFN-γ to mediate efficient and more-effective parasite control in mice infected with L. major (41). Although not investigated in this current study, it is conceivable that deficiency of Sema3E leads to increased IL-17 response following L. major infection. This increased IL-17 response could possibly synergize with increased IFN-γ response, resulting in enhanced resistance to L. major.
In conclusion, we have demonstrated that Sema3E enhances susceptibility to L. major infection. Sema3E-deficient mice displayed enhanced resistance to L. major infection, and this was associated with increased CD11C+ cell activation and production of IL-12p40, leading to a concomitant increase in Th1 immune response. To the best of our knowledge, this is the first report showing the role of Sema3E in the pathogenesis of cutaneous leishmaniasis in particular and protozoan infections in general. Because Sema3E is a negative regulator of the host immune response to L. major infection, immunotherapeutic strategies involving neutralization of Sema3E in an infected individual may result in better disease outcomes.
We thank the members of the Parasite Vaccines Development Laboratory for technical assistance during this study. Some figure elements were created using Biorender.com.
This work was supported by a grant from the Canadian Institutes of Health Research (MOP 114932) and Research Manitoba.
The online version of this article contains supplemental material.
Abbreviations used in this article:
bone marrow–derived CD11c+ cell
bone marrow–derived macrophage
soluble Leishmania Ag
regulatory T cell
The authors have no financial conflicts of interest.