Brugia malayi Microfilariae Induce a Regulatory Monocyte/Macrophage Phenotype That Suppresses Innate and Adaptive Immune Responses

Background Monocytes and macrophages contribute to the dysfunction of immune responses in human filariasis. During patent infection monocytes encounter microfilariae in the blood, an event that occurs in asymptomatically infected filariasis patients that are immunologically hyporeactive. Aim To determine whether blood microfilariae directly act on blood monocytes and in vitro generated macrophages to induce a regulatory phenotype that interferes with innate and adaptive responses. Methodology and principal findings Monocytes and in vitro generated macrophages from filaria non-endemic normal donors were stimulated in vitro with Brugia malayi microfilarial (Mf) lysate. We could show that monocytes stimulated with Mf lysate develop a defined regulatory phenotype, characterised by expression of the immunoregulatory markers IL-10 and PD-L1. Significantly, this regulatory phenotype was recapitulated in monocytes from Wuchereria bancrofti asymptomatically infected patients but not patients with pathology or endemic normals. Monocytes from non-endemic donors stimulated with Mf lysate directly inhibited CD4+ T cell proliferation and cytokine production (IFN-γ, IL-13 and IL-10). IFN-γ responses were restored by neutralising IL-10 or PD-1. Furthermore, macrophages stimulated with Mf lysate expressed high levels of IL-10 and had suppressed phagocytic abilities. Finally Mf lysate applied during the differentiation of macrophages in vitro interfered with macrophage abilities to respond to subsequent LPS stimulation in a selective manner. Conclusions and significance Conclusively, our study demonstrates that Mf lysate stimulation of monocytes from healthy donors in vitro induces a regulatory phenotype, characterized by expression of PD-L1 and IL-10. This phenotype is directly reflected in monocytes from filarial patients with asymptomatic infection but not patients with pathology or endemic normals. We suggest that suppression of T cell functions typically seen in lymphatic filariasis is caused by microfilaria-modulated monocytes in an IL-10-dependent manner. Together with suppression of macrophage innate responses, this may contribute to the overall down-regulation of immune responses observed in asymptomatically infected patients.


Introduction
Lymphatic filariasis is an immune-mediated spectral disease that manifests in two main clinical outcomes: chronic pathology or asymptomatic infection. These outcomes depend on a multitude of factors, including parasite-induced immunoregulation and host genetic background (reviewed elsewhere [1]).
An overt manifestation is the hyperresponsive phenotype that develops in patients with chronic lymphatic pathology (abbreviated as CP). These individuals have increased antigen-specific immunoglobulin (Ig)E and low IgG4 [2,3], strong T helper (Th)1 and Th17 proinflammatory responses and a greatly diminished T regulatory (Treg) compartment [2], resulting in immunopathological changes in the host. CP patients carry adult worms in the lymphatics but are generally amicrofilaremic, as a strong immune response kills the microfilarial stage. Parasite death leads to the release of antigenic material that triggers inflammation and causes destruction of lymphatic vessels and inflammation [4]. In Wuchereria bancrofti, Brugia malayi and B. timori infections, this can result in the development of elephantiasis or hydrocoele, whereby the lymphatic tissue becomes dilated and hypertrophic.
The second clinical manifestation is a hyporesponsive phenotype characterised by asymptomatic infection (abbreviated as AS), which tolerates the presence of fecund adult worms due to strong parasite-induced immunosuppression and immunomodulation [5]. Importantly, adult worms are tolerated and circulating blood microfilariae are carried by these patients, ensuring transmission. This group has increased numbers of regulatory cells, high interleukin (IL)-10 and elevated levels of antigen-specific IgG4 leading to a modified Th2 response that protects the host and permits parasite survival [5]. Thus, parasite-induced immunomodulation allows persistent infection and continuous transmission while simultaneously enabling the host to tolerate infection by diminishing clinical symptoms. The proportion of individuals in a filaria-endemic area who do not develop one of these two clinical manifestations remain infection-and disease-free and are putatively immune; these individuals are known as endemic normals (abbreviated as EN) [1].
Filaria have been shown to act on host dendritic cells, monocytes, macrophages, T cells and B cells to cause immunomodulation, typically inducing Th2 type and regulatory responses [6]. Immunomodulation occurs through production of specific parasite-derived products that target mammalian host immune cells and signalling pathways. This is strictly dependent on live parasites as shown by the recovery of cellular responsiveness in patients treated with microfilaricidal chemotherapy, specifically diethylcarbamazine (DEC) [7]. While some adults are killed by DEC treatment, the main target is the microfilarial stage, suggesting a prominent role for microfilariae in modulating immune responses [8].
Monocytes and macrophages have long been described to develop a particular phenotype in filarial infection that may contribute to the dysfunction of adaptive immune responses. The spectrum of phenotypes and functions that monocytes and macrophages can develop is vast, and ranges from classical activation, through to alternative activation, wound healing, parasite resistance and immune regulation (reviewed elsewhere [9]). Thus classical activation is characterised by production of proinflammatory molecules and typically develops in response to stimulation with lipopolysaccharide (LPS) plus a secondary stimulus such as interferon (IFN)-c or tumour necrosis factor (TNF)-a. In contrast alternative activation is induced by stimulation with IL-4 and/or IL-13 and induces macrophages that are involved in wound healing and immune regulation [9]. Murine macrophages that develop in response to these cytokines express specific markers, including arginase (arg)-1, resistin-like molecule (RELM)-a, Ym-1, Ym-2, acidic mammalian chitinase (AMCase), and mannose receptor C type (MRC)-1 [9]. This is a phenotype that is reflected in macrophages in filarial infections [10]. In contrast to the murine system, human monocytes and macrophages typically show a diverse phenotype after stimulation with IL-4, defined by expression of the scavenger receptor CD163 and the chemokine ligand CCL18 [11,12]. MRC-1 (CD206), similar to mice, is also expressed by human macrophages in this setting [12]. In human filarial infections, expression of arg-1, programmed death-ligand (PD-L)1 and PD-L2 on monocytes has also been reported [13,14].
Macrophages recruited in filarial infection induce specific hyporesponsiveness in T cells [15,16]. Macrophages recruited during B. malayi infection drive CD4 + Th2 responses, deviating the immune system from inducing a proinflammatory Th1 response that could be detrimental to parasite survival [17]. During patent filarial infection monocytes encounter the microfilarial lifecycle stage in the blood, before migrating out to the tissues. This initial contact with the parasite is particularly interesting as it occurs only in AS patients where the adult worms are tolerated in the lymphatics and produce viable microfilariae [18].
We hypothesised that microfilariae in circulation act on human monocytes during patent infection. This primary contact may affect monocyte differentiation, macrophage development and the ensuing innate and adaptive responses and thus may contribute to the development and maintenance of asymptomatic infection. Therefore we aimed to characterise the phenotype of monocytes and macrophages stimulated in vitro with B. malayi microfilarial (Mf) lysate, and to determine the effect of B. malayi Mf lysatestimulated monocytes and macrophages on defined innate or adaptive functions. We could show that monocytes stimulated with B. malayi Mf lysate in vitro develop a defined regulatory phenotype, characterised by expression of the immunoregulatory markers IL-10 and PD-L1. Significantly, this regulatory phenotype could be recapitulated in monocytes from Wuchereria bancrofti AS patients in contrast to CP patients and EN individuals. Monocytes from nonendemic normal donors stimulated with Mf lysate directly inhibited CD4 + T cell proliferation and cytokine production (IFN-c, IL-13 and IL-10). Importantly, IFN-c responses could be restored by neutralisation of IL-10 or PD-1. Furthermore, macrophages stimulated with Mf lysate expressed high levels of IL-10 and had suppressed phagocytic abilities. Finally we could show that Mf lysate applied during the differentiation process of macrophages in vitro interfered with macrophage abilities to respond to subsequent LPS stimulation in a selective manner.

Ethics statement
All experiments with material from filaria non-endemic normal donors were approved by the ethical committee of the Charité,

Author Summary
Lymphatic filariasis is a parasitic disease that affects over one million people worldwide, causing chronic morbidity in the majority of infected individuals. A certain proportion of individuals develop asymptomatic infection that allows persistence of the parasite and therefore transmission of disease through the blood-circulating microfilariae. We show that monocytes and macrophages, innate effector cells that circulate in the blood, migrate or reside in tissues and come into contact with microfilariae, can be stimulated by microfilarial (Mf) lysate in vitro to develop a regulatory phenotype via expression of PD-L1 and IL-10. Significantly, this regulatory monocyte phenotype was directly reflected in monocytes isolated from filaria asymptomatically infected patients in the absence of external stimuli. Mf lysate-modulated monocytes inhibited adaptive immune functions, some of which could be restored by neutralisation of IL-10. Mf lysate-modulated macrophages had reduced phagocytic capacity, while macrophages differentiated in the presence of Mf lysate displayed significantly inhibited innate responses to LPS stimulation. This suggests that microfilariae modulate the innate response by acting on macrophage differentiation and macrophages themselves, and modulate the adaptive CD4 + T cell response by acting on monocytes. The phenotypic pattern observed by modulated monocytes is recapitulated in vivo in active infection. This highlights a previously unclear mechanism of immune modulation by the parasite.
Berlin (permit number EA1/104/14). All experiments with material from W. bancrofti-exposed donors were approved by the ethical committee of the Blue Peter Public Health and Research Center-LEPRA Society, Hyderabad (permit number 5/2009). Informed written consent was obtained from all participants. All W. bancrofti infected donors were treated for lymphatic filariasis by administration of DEC and symptomatic relief after completion of the study. The study was performed according to the Declaration of Helsinki.

Filarial lysate preparation
Live B. malayi microfilariae and adult female worms were a kind donation from the NIAID/NIH Filariasis Research Reagent Resource Center (FR3) in Athens, Georgia. Microfilariae and adult female worms were washed twice in RPMI medium containing 200 U/ml penicillin and 200 mg/ml streptomycin. To collect excretory/secretory (ES) products, microfilariae were cultured for 3-5 days in RPMI containing 1% glucose, 200 U/ml penicillin and 200 mg/ml streptomycin in a 5% CO 2incubator at 37uC in 6 well plates whereby media was replaced every 24 h. The resulting ES-containing media was concentrated using Vivacell 70 concentrators (Sartorius Stedim Biotech GmbH, Göttingen, Germany) with a membrane cut-off at 5,000 molecular weight. To prepare microfilarial (Mf) lysate, live microfilariae in suspension were subsequently harvested, washed twice in phosphate buffered saline (PBS) by centrifuging for 10 min at 1500 rpm. Pelleted microfilariae or adult female worms were homogenised directly in a glass homogeniser and ultrasonicated on ice at an intensity of 10% for 3 min. The homogenate was centrifuged at 12,000 rpm and 4uC for 10 min and sterile filtered through a 0.22 mm filter. Protein concentration was determined using the Pierce BCA protein assay kit (Thermo Scientific, Waltham, USA) as per the manufacturer's guidelines. LPS concentration was determined by Limulus amoebocyte lysate endotoxin detection kit QCL-1000 (Lonza, Walkersville, USA); the LPS content in B. malayi Mf lysate, ES or adult female (Fem) lysate used in all assays was ,1 EU/ml in the final concentration.

Human study populations
In vitro experiments using samples from filaria non-endemic normal donors were performed in Germany, using buffy coats purchased from the German Red Cross. Experiments using samples from filaria-exposed donors examined a cohort of 56 individuals from Andhra Pradesh in South India, where lymphatic filariasis caused by W. bancrofti is endemic. Night blood smears were performed with 20 ml blood to detect circulating microfilariae, and the TropBio Og4C3 ELISA (TropBio Pty. Ltd, Townsville, Queensland, Australia) was performed using serum to detect circulating filarial antigen (CFA), as per the manufacturer's instructions. Patients with lymphatic filarial pathology (lymphadenitis, lymphoedema, hydrocoele) were examined as part of a clinical protocol approved by the institutional ethical committee of the Blue Peter Public Health and Research Center-LEPRA Society. Based on these results, 28 individuals were classed as endemic normals (EN), 21 had chronic pathology (CP) and 7 had asymptomatic infection (AS) ( Table 1). Any AS patient found to be positive for CFA was classed as asymptomatic regardless of the night blood smear result; at 20 ml blood per smear, the test has low sensitivity, giving a cut off value of 50 microfilariae per ml.

Isolation of peripheral blood mononuclear cells
For in vitro experiments using samples from non-endemic normal donors, peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats. For experiments using samples from filaria-exposed donors, PBMCs were isolated from 40 ml blood. In both cases PBMCs were isolated by density centrifugation using Lymphocyte Separation Medium (LSM). Blood from buffy coats was initially diluted in phosphate buffered saline (PBS), two parts blood to one part PBS. Briefly, blood was layered onto LSM and centrifuged at 2500 rpm, at room temperature for 25 min with zero brake and zero accelerator. The interphase was collected and washed in PBS plus 0.2% bovine serum albumin (BSA). Cells were centrifuged at 1500 rpm, 4uC for 10 min, washed in PBS plus 0.2% BSA and centrifuged at 1000 rpm, 4uC for 10 min to remove platelets. Lysis of erythrocytes was performed to remove remaining erythrocytes if necessary. For this, 5 ml of ammoniumchloride-potassium lysis buffer (0.01 M KHCO 3 , 0.155 M NH 4 Cl, 0.1 mM EDTA, pH 7.5) was added to cells for 5 min at room temperature, after which cells were washed in PBS plus 0.2% BSA and centrifuged at 1500 rpm for 10 min.

Isolation of CD14 + monocytes and differentiation to macrophages
To positively select for CD14 + monocytes, anti-CD14 beads (Miltenyi, Biotec, Bergisch-Gladbach, Germany) were added for 20 min to PBMCs. A volume of 200 ml beads was used on buffy coats, while 50 ml was used on 40 ml whole blood. In both cases, cells were washed in PBS plus 0.2% BSA, 2 mM EDTA and filtered before separation using a 70 mm filter (Partek, St. Louis, USA). Cells from non-endemic normal donors were separated by an autoMACS classic, using the program 'possel' (Miltenyi Biotec, Bergisch-Gladbach, Germany). Cells from filaria-exposed donors were separated using MACS MS columns as per the manufacturer's instructions (Miltenyi Biotec, Bergisch-Gladbach, Germany). After isolation, cells were washed once in PBS plus 0.2% BSA, 2 mM EDTA, centrifuged at 1500 rpm and 4uC for 5 min and transferred into complete RPMI medium (RPMI 1640, 5% AB human serum, 100 U/ml penicillin, 100 mg/ml streptomycin, 1 mM L-glutamine, 1 mM MEM non-essential amino acids, 1 mM sodium pyruvate) for use in subsequent experiments. Macrophages were generated in vitro by culturing CD14 +

In vitro stimulation of monocytes and macrophages
Monocytes from filaria-exposed donors were used directly for ex vivo RT-PCR analysis or seeded at a concentration of 0.2610 6 per well into 96-well flat bottom cell culture plates and stimulated for 24 h in vitro with 200 ml stimulus. Monocytes from non-endemic normal donors were seeded at a concentration of 2610 6 per well into 24-well cell culture plates and stimulated for 4 h or 24 h in vitro with 1 ml stimulus. In vitro generated macrophages were washed on day 6 with PBS, and the culture supernatant was replaced with 1 ml stimulus for 24 h. Cells were stimulated with 20 ng/ml IL-4 (Peprotech, Rocky Hill, USA), a combination of 20 ng/ml IFN-c (Peprotech, Rocky Hill, USA) plus 100 ng/ml LPS (Invivogen, California, USA) or 20 mg/ml B. malayi Mf lysate at 37uC and 5% CO 2 . Unstimulated controls were included in all experiments. The supernatant was collected and stored at 220uC for further analysis. Cells were lysed in RNA lysis buffer (Analytik Jena, Jena, Germany) for RT-PCR analysis.

Cytokine analysis
IL-6, IL-8, IL-10, IL-13, IFN-c and TNF-a protein were measured using a commercial enzyme-linked immunosorbent assay (ELISA) kit from eBioscience (San Diego, USA). IL-12p40 was measured using an ELISA kit from BioLegend (San Diego, USA). All samples were measured in duplicates. Absorbance was read at 450 nm with background wavelength subtracted at 570 nm using the Synergy HT plate reader from BioTek (Winooski, USA).
Active transforming growth factor (TGF)-b was measured using a TGF-b reporter cell line. Briefly, MFB-F11 cells were incubated for 24 h with cell culture supernatant. The resulting supernatant was assessed using the SEAP Reporter Gene Assay (Roche, Branford, USA) according to the manufacturer's instructions.

RNA extraction and real-time PCR
RNA was isolated from cells using a commercial kit, following the manufacturer's instructions (innuPREP RNA mini-kit, Analytik Jena, Jena, Germany). The concentration of extracted RNA was determined using the NanoDrop 1000. RNA was reversetranscribed to cDNA using a high capacity RNA-to-cDNA kit (Life Technologies, Darmstadt, Germany). cDNA was set to a concentration of 3-10 ng/ml, depending on the experiment. Realtime PCR was performed with FastStart Universal SYBR Green Master Mix (Roche Applied Science, Indianapolis, USA) using the ABI 7300 Real-Time PCR (Life Technologies, Darmstadt, Germany). Relative changes in gene expression were calculated with ABI 7300 SDS Software (Life Technologies, Darmstadt, Germany). For monocytes and macrophages from non-endemic normal donors, expression levels of transcripts were normalized to the Ct values of the endogenous housekeeping gene by using the 2 2DDCt method [19]. Relative expression of genes in stimulated samples was compared to unstimulated controls (which are set at 1). For monocytes from filarial-exposed donors we chose not to normalise data to the reference group (EN), as there was a large variation in Ct values within the heterogeneous EN group. Thus expression levels of transcripts were normalized to the Ct values of the endogenous housekeeping gene by using the method 1/2 DCt where DCt represents the difference between the target gene and the housekeeping gene. Baseline expression of samples from AS or CP was compared to that of EN. In all experiments ß2microglobulin was used as a housekeeping gene. The gene-specific primer sequences and accession numbers are shown in Table S1.

Isolation of CD4 + T cells and CFSE labelling
PBMCs were labelled using the CD4 + T cell Isolation Kit II (Miltenyi Biotec, Bergisch-Gladbach, USA) according to the manufacturer's instructions and sorted on an autoMACS classic (Miltenyi Biotec, Bergisch-Gladbach, USA) using the program 'deplete'. Untouched CD4 + T cells were stained with CFSE and rested in complete RPMI for 24 h at 37uC and 5% CO 2 .

Monocyte: CD4 + T cell neutralisation assay
To determine a role for IL-10 signalling or PD-1-PD-L1 interactions in the suppression of CD4 + T cell effector functions induced by Mf lysate, monocytes were left unstimulated or stimulated with 20 mg/ml B. malayi Mf lysate for 24 h in a 5% CO 2 -incubator at 37uC in 6-well plates. Monocytes were washed and 1610 5 monocytes were cocultured with 5610 5 CFSE-labelled CD4 + T cells in 96-well plates in the presence of 2 mg/ml soluble anti-CD3 (OKT3, eBioscience, San Diego, USA) plus neutralising anti-IL-10 antibodies or anti-PD-1 antibodies (both at 10 mg/ml, from eBioscience San Diego, USA). After 3-5 days, the supernatant was removed for cytokine analysis and cells were stained with Fixable Viability Dye eFluor 780 (eBioscience, San Diego, USA) and anti-CD4-PE-Cy5 (clone RPA-T4, BioLegend, San Diego, USA). Fixed cells were acquired using the FACSCanto II (BD, Franklin Lakes, USA) and analysed using FlowJo, version 8.8.7 (Tree Star, Ashland USA).

LPS stimulation of Mf lysate-differentiated macrophages
To determine the ability of Mf lysate-differentiated macrophages to respond to LPS stimulation, Mf lysate-differentiated macrophages were washed on day 6 of culture and 2610 5 cells were stimulated for 24 h with 100 ng/ml LPS (Invivogen, California, USA). The following day supernatants were collected for cytokine analysis by ELISA.

Phagocytosis assay
Macrophages were generated in vitro as above, and used after 6 days incubation. To harvest macrophages, the cells were washed three times in PBS, then 1 ml PBS containing 5 mM EDTA was added. The cells were kept at 4uC for 10 min then scraped off the well using a cell scraper. Macrophages were seeded into a 96-well flat bottom plate (0.2610 6 cells per well) in complete RPMI, and stimulated using 20 mg/ml B. malayi Mf lysate for 24 h. After 24 h macrophages were washed in PBS three times, after which the prepared fluorescent pHrodo BioParticles suspension was added. pHrodo BioParticles (Life Technologies, Darmstadt, Germany) were prepared beforehand by suspending 2 mg BioParticles in 2 ml of uptake buffer (140 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl 2 , 1.0 mM MgCl 2 , 20 mM HEPES, pH 7.4). The solution was briefly vortexed and then sonicated for 5 min to ensure homogenous dispersal of the particles. 100 ml of the prepared suspension was then added to the cells. Unstimulated macrophages were regarded as a positive control. Cytochalasin D (20 mM) was added as a negative control to unstimulated cells. Cells were incubated with the fluorescent particles for 3 h at 37uC (no CO 2 ). Finally the cells were washed three times in PBS and 200 ml of 0.5% formalin was added. Fluorescence was read at 550 nm excitation and 600 nm emission using the Synergy HT plate reader from BioTek (Winooski, USA). The net phagocytosis was calculated as per the manufacturer's instructions, by subtracting the average fluorescence intensity of the negative control from the positive control and all experimental wells. The phagocytosis response to the experimental effector (% phagocytosis) could then be calculated as a percentage of the net positive control phagocytosis (% phagocytosis = net phagocytosis6100/net phagocytosis of positive control.

Statistical analysis
All statistical analyses were performed using GraphPad Prism version 6.0d (GraphPad Software, Inc., California, USA). In experiments where two paired groups were analysed, Wilcoxon signed-rank test was used to compare a condition to its unstimulated control. In experiments where more than two paired groups were analysed, Friedman's ANOVA was used to determine whether a statistically significant difference existed between any of the conditions and the unstimulated control (significance level p, 0.05). In case of significance, the main analysis was followed up with a Wilcoxon signed-rank test between a condition and the unstimulated control, whereby a Bonferroni correction was applied. The Kruskal-Wallis test with Dunn's multiple comparisons post-test was used to determine statistical significance between multiple unpaired groups.

Results
Monocytes stimulated in vitro with B. malayi Mf lysate develop a specific activation phenotype To understand if B. malayi microfilariae act on monocytes to induce immune modulation, monocytes isolated from buffy coats from filaria non-endemic normal donors were stimulated for 24 h in vitro with B. malayi Mf lysate, LPS plus IFN-c or IL-4 (Fig. 1).
Monocytes stimulated with B. malayi Mf lysate produced significant and high levels of IL-10, IL-6, TNF-a and IL-8 while IL-12p40 was not induced (Fig. 1A). IL-27 was not detected (data not shown). Control stimulation with LPS plus IFN-c resulted in significantly elevated levels of IL-10, IL-6, TNF-a, IL-8 as well as IL-12p40, which was in stark contrast to Mf lysate-stimulated monocytes. On the other hand, control stimulation with IL-4 significantly inhibited protein production of IL-6 and IL-8 compared to unstimulated controls, while levels of IL-10, TNF-a and IL-12p40 were not significantly altered from unstimulated controls (Fig. 1A). Using a TGF-b reporter cell line, the levels of active TGF-b were measured (Fig. 1B). Stimulation of monocytes with B. malayi Mf lysate, LPS plus IFN-c or IL-4 did not alter TGF-b production compared to unstimulated controls.
To assess the expression in monocytes of markers associated with an alternative/regulatory phenotype, mRNA expression of MRC-1 (NM_002438), CCL18 (NM_002988), PD-L1 (NM_014143) and PD-L2 (NM_025239) was analysed after 24 h stimulation (Fig. 1C). B. malayi Mf lysate induced significantly higher levels of CCL18 and PD-L1 compared to unstimulated controls, while the other markers were not altered. Monocytes stimulated with LPS plus IFN-c or IL-4 upregulated expression of CCL18, PD-L1 and PD-L2 compared to unstimulated controls. MRC-1 was significantly inhibited by LPS plus IFN-c compared to unstimulated controls.
To determine whether the high mRNA level of PD-L1 was reflected on a protein level, surface expression of PD-L1 was measured by flow cytometry. Expression of the activation markers HLA-DR, CD80 and CD86, the human monocyte alternatively activated markers PD-L2, CD163 and CD206 and the classical activation marker IDO was measured in parallel (Fig. 1D). In agreement with the PCR data, PD-L1 was significantly upregulated in monocytes in response to B. malayi Mf lysate stimulation (Fig. 1D). Significant upregulation of PD-L2 was also observed, however HLA-DR, CD80, CD86, CD163, CD206 and IDO were not significantly different from unstimulated controls. LPS plus IFN-c induced expression of HLA-DR, PD-L1 and PD-L2, while stimulation with IL-4 upregulated CD86, PD-L1 and PD-L2 (Fig. 1D). The other markers analysed were not altered after stimulation with LPS plus IFN-c or IL-4.
It has previously been shown that microfilariae affect the survival of dendritic cells by apoptosis [20,21]. To determine whether B. malayi Mf lysate affected cell viability, monocytes, in vitro generated macrophages or macrophages differentiated in the presence of Mf lysate were stained with a dead cell exclusion dye (Fig. S1) and viability was assessed by flow cytometry. There was no difference in the percentage of viable cells in B. malayi Mf lysate-stimulated versus unstimulated cells.

Monocytes from W. bancrofti asymptomatically infected donors have a regulatory phenotype at baseline
Stimulation in vitro with B. malayi Mf lysate induced a defined regulatory phenotype of monocytes that significantly upregulated the proinflammatory markers IL-6, TNF-a and IL-8 as well as the alternative/regulatory markers IL-10 and PD-L1 in monocytes. Therefore we hypothesised that in filaria-infected individuals, monocytes from asymptomatically infected patients, who exhibit circulating microfilariae in their blood, develop a phenotype similar to that observed in our in vitro experiments. Thus, we performed RT-PCR on monocytes isolated from PBMCs from EN, CP and AS donors (Fig. 2). There was a trend for monocytes from AS patients to express elevated levels of the alternative/ regulatory markers IL-10 (NM_000572), MRC-1, CCL18, PD-L1 and PD-L2 as well as the proinflammatory markers IL-6 (NM_000600), TNF-a (NM_000594) and IL-12p40 (NM_002187). IL-8 (NM_000584) was significantly downregulated in AS patients compared to EN individuals. MRC-1 was significantly elevated in AS patients compared to CP donors. In contrast, there appeared to be little difference between CP and EN although IL-10 and IL-6 were significantly downregulated in CP compared to EN donors. Thus, monocytes isolated from PBMCs from AS patients recapitulate to a great extent the expression profile observed in B. malayi Mf lysate-stimulated monocytes from filaria non-endemic donors.
Monocytes from AS patients have been shown to be functionally defective in terms of Toll-like receptor (TLR) expression and function [22,23]. Thus we wanted to establish the capacity of monocytes from AS patients to respond to B. malayi Mf lysate (Fig. S2). After 24 h without stimulation in culture, monocytes from EN, CP and AS donors responded equally in terms of protein production of IL-10, IL-6, TNF-a and IL-12p40. After 24 h stimulation with B. malayi Mf lysate, monocytes from all three groups responded by producing equal amounts of cytokines. Hence, there was no inherent defect in the ability of monocytes from EN, CP and AS donors to produce cytokines in response to B. malayi Mf lysate.

B. malayi Mf lysate-stimulated monocytes impair CD4 + T cell proliferation and cytokine production
To determine whether the activation phenotype seen in monocytes stimulated with B. malayi Mf lysate may account for the hyporesponsiveness of CD4 + T cells observed in ex vivo studies with PBMCs from AS patients [18,[24][25][26][27] a coculture assay with autologous CD4 + T cells was employed. To this end, autologous CFSE-labelled CD4 + T cells were polyclonally stimulated and incubated with B. malayi Mf lysate-stimulated monocytes (Fig. 3). T cell proliferation was significantly inhibited after coculture with B. malayi Mf lysate-stimulated monocytes (Fig. 3A). The production of IFN-c, IL-13 and IL-10  was significantly inhibited in the supernatant of cocultures with B. malayi Mf lysate-stimulated monocytes compared to cocultures with unstimulated monocytes (Fig. 3B). Thus, Mf lysatemodulated monocytes showed a significantly impaired ability to stimulate CD4 + T cell functions.
In order to understand if monocytes treated with microfilariaderived ES products could alter T cell responses we repeated the coculture but stimulated monocytes with 20 mg/ml B. malayi microfilarial ES. Microfilarial ES-stimulated monocytes did not suppress CD4 + T cell effector functions (Fig. S3).
Neutralisation of IL-10 and PD-1 restores CD4 + T cell IFN-c production As IL-10 and PD-L1 were significantly upregulated in B. malayi Mf lysate-stimulated monocytes, we hypothesised that one of these molecules could be involved in inhibiting CD4 + T cell functions as observed in Fig. 3. Thus we repeated the coculture experiment and included neutralising anti-IL-10 antibodies to block IL-10 signalling or anti-PD-1 antibodies to block PD-1-PD-L1 interactions (Fig. 4). Experiments using unstimulated monocytes in the coculture were performed as a control (Fig. S4). While proliferation of T cells was elevated after neutralisation of IL-10 to a statistically significant level (p = 0.016, Fig. 4A), the biological difference in restoration was minimal (44.78% of T cells proliferated in response to Mf lysatestimulated monocytes compared with 48.59% of T cells proliferating after IL-10 was neutralised). Proliferation of T cells was not changed after neutralisation of PD-1 (Fig. 4B). IFN-c production was restored in response to neutralisation of IL-10 ( Fig. 4A) and PD-1 (Fig. 4B), while IL-13 responses were not restored.
To determine whether monocytes were actively expressing IL-10 upon the time of coculture, B. malayi Mf lysate-stimulated monocytes were assessed for IL-10 mRNA expression by RT-PCR (Fig. S5). After 24 h stimulation with B. malayi Mf lysate, there was a trend for monocytes to express IL-10 mRNA, although this did not reach statistical significance.

Macrophages stimulated in vitro with B. malayi Mf lysate develop a specific activation phenotype
To understand if B. malayi microfilariae act on macrophages to induce immune modulation, monocytes isolated from buffy coats from filaria non-endemic normal donors were differentiated to macrophages in vitro and stimulated for 24 h in vitro with B. malayi Mf lysate, LPS plus IFN-c or IL-4 (Fig. 5). Similar to monocytes (see Fig. 1A), stimulation with B. malayi Mf lysate led macrophages to produce significantly higher levels of IL-10 and IL-8 compared to unstimulated controls, while IL-6, TNF-a and IL-12p40 were not induced (Fig. 5A). IL-27 was not detected (data not shown). This is in clear contrast to stimulation with LPS plus IFN-c, which led to significant and high expression of IL-10, IL-6, TNF-a, IL-12p40 and IL-8. IL-4 stimulation did not induce significant production of any cytokines measured (Fig. 5A). Using a TGF-b reporter cell line, the levels of active TGF-b were measured (Fig. 5B). Stimulation of macrophages with B. malayi Mf lysate, LPS plus IFN-c or IL-4 did not alter production of TGF-b compared to unstimulated controls. On mRNA level, macrophages stimulated with B. malayi Mf lysate did not alter expression of MRC-1, CCL18, PD-L1 or PD-L2 compared to unstimulated controls (Fig. 5C). There were no significant changes in expression of these markers after stimulation of macrophages with LPS plus IFN-c or IL-4 compared to unstimulated controls (Fig. 5C).
In terms of cell surface markers, stimulation with B. malayi Mf lysate did not alter expression of HLA-DR, CD80, CD86, PD-L1 or PD-L2 in macrophages compared to unstimulated controls (Fig. 5D). A similar result was observed in macrophages stimulated with LPS plus IFN-c or IL-4 (Fig. 5D).

B. malayi Mf lysate interferes with the differentiation of macrophages in vitro
It is feasible that microfilariae interfere with the differentiation of monocytes to macrophages in vivo during patent infection, due to their shared anatomical locations. Thus we determined the phenotype and functions of macrophages generated in vitro from CD14 + monocytes in the presence of 20 mg/ml B. malayi Mf lysate (Fig. 6). Mf lysate-differentiated macrophages did not alter expression of the maturation markers HLA-DR, CD80 and CD86 or the macrophage markers CD11b and CD163 compared to macrophages generated in the absence of Mf lysate (Fig 6A). However when macrophages generated in the presence of Mf lysate were washed and stimulated with 100 ng/ml LPS, there was a significant and selective inhibition of IL-6, TNF-a and IL-12p40 but not IL-10, when compared to macrophages generated in the absence of Mf lysate (Fig. 6B).

B. malayi Mf lysate-stimulated macrophages display impaired phagocytosis
To determine whether phagocytosis of macrophages was modulated by stimulation with B. malayi Mf lysate, in vitro generated macrophages were stimulated for 24 h with B. malayi Mf lysate. Subsequently the phagocytic capacity was determined by measuring the phagocytosis of fluorescently labelled bioparticles, whereby unstimulated cells were used as a positive control (Fig. 6C). B. malayi Mf lysate significantly inhibited phagocytosis in macrophages, reducing this function by approximately 20%.

Discussion
Our data show that B. malayi Mf lysate induces a regulatory monocyte phenotype that curtails CD4 + T cell effector functions in vitro. This monocyte population is characterized by expression of IL-10 and PD-L1 as well as certain proinflammatory markers. This regulatory phenotype is reflected in monocytes from AS patients with active filarial infection, but not in CP patients or EN donors. Importantly, CD4 + T cell IFN-c responses could be recovered after neutralisation of IL-10 or PD-1. Furthermore we could show that macrophages stimulated with Mf lysate expressed high levels of IL-10 and had suppressed phagocytic abilities. Finally, Mf lysate applied during the differentiation process of macrophages in vitro interfered with macrophage abilities to respond to subsequent LPS stimulation in a selective manner. A limitation to our results from filaria-exposed donors was the access to only very low numbers of AS donors used for analysis of ex vivo monocyte phenotype and for stimulation of filaria-endemic monocytes in vitro. Such low numbers of AS patients are in part due to the extensive mass drug treatment effort in South India during the last decade [28,29].
The observed monocyte phenotype is in accordance with a previous study that described monocytes stimulated with live microfilariae to upregulate PD-L1 and to have an alternatively activated phenotype [14]. Monocytes isolated from filarial lysatetreated PBMCs from humans with asymptomatic filarial infection are characterised by increased expression of arg-1 and IL-10 and decreased levels of nitric oxide synthase (NOS, typically used as a classical activation marker in murine studies) compared to endemic normal controls [13]. Interestingly in our studies inducible NOS (iNOS) could not be detected in B. malayi Mf lysate-treated monocytes or macrophages in vitro or in monocytes from endemic patients ex vivo (data not shown). iNOS historically can be difficult to detect, thus the differences observed here may lie in the techniques used [30]. Furthermore murine markers cannot simply be translated into the human system [31,32]. As an example, reports indicate that arg-1 may not be a reliable marker for alternatively activated monocytes or macrophages in humans as it is found in other cell types [33]. Human monocytes do not express arg-1 after stimulation with IL-4 and IL-13 (the prototypical inducers of alternative activation), unlike mouse macrophages [31]. Thus we have carefully selected a number of markers that have previously been reported for classical activation, alternative activation, or regulation in human monocytes and macrophages [9,11,12,31,34]. We have used these as the basis for characterising the cells in the current study.
Our results indicate a phenotype that consists of a spectrum of proinflammatory, alternatively activated and regulatory markers, which is in line with numerous reports stating the mixed Th1/Th2 response that is often seen after exposure to microfilariae [18,20,[35][36][37]. The mixture of markers observed may be partly influenced by Wolbachia, the obligate endosymbiont found in all lifecycle stages of the filarial parasites W. bancrofti and B. malayi. Wolbachia are known to induce inflammatory responses in monocytes in a TLR4-independent manner [38][39][40]. Contrary to this, monocytes treated with lysate from adult female worms do not upregulate any of the cytokines tested. This highlights a microfilaria-derived factor other than Wolbachia to be responsible for the effects seen here with Mf lysate-treated monocytes (Fig.  S6). Importantly in our studies, Mf lysate did not induce production of IL-12p40, in stark contrast to stimulation of monocytes or macrophages with LPS plus IFN-c. Similarly there was no detection of TNF-a in Mf lysate-stimulated macrophages, which was highly induced by LPS plus IFN-c. Monocytes showed some production of TNF-a however this was approximately five times less than that observed after stimulation with LPS plus IFNc. On the other hand, the proinflammatory markers as well as IL-10 were not detected in either monocytes or macrophages after stimulation with IL-4. Thus Mf lysate produces a clear and distinct response to that induced by the hallmark proinflammatory stimulus, LPS plus IFN-c or the prototypical alternative activation stimulus, IL-4.
Nevertheless it is unclear to what extent the expression of proinflammatory cytokines may function in the face of high levels of immunoregulatory cytokines such as IL-10. We have previously shown that IL-10 is induced in murine macrophages by a nematode-derived cysteine-proteinase inhibitor isolated from a related filarial species, Acanthocheilomena viteae (AvCystatin) [41]. Thus it would be interesting to investigate the role of filarial cystatin in B. malayi Mf lysate-induced IL-10 expression, as B. malayi contains three different forms of cystatin, with Bm-CPI-2 being more closely related to AvCystatin [42]. Furthermore, AvCystatin was shown to mediate IL-10-and macrophagedependent immunomodulation in a mouse model of airway hyperreactivity [43]. Thus, future experiments should determine the contribution of B. malayi cystatins to human T cell hyporesponsiveness.
Establishing the phenotype and function of B. malayi Mf lysatestimulated monocytes and macrophages from non-endemic normal donors highlights these cells as instruments of microfilarial immune modulation. To elucidate the exact phenotype of monocytes during infection, it was necessary to analyse the cytokine and marker profile of monocytes from endemic individuals ex vivo, without prior stimulation in the presence of other immune cells as done in other studies [13]. Therefore we examined monocytes from individuals with W. bancrofti asymptomatic infection that had presumably interacted with microfilariae in circulation in the 12 hours prior to isolation. As expected, in the absence of any external stimulation, only monocytes from this group produced the specific phenotype that was previously observed in vitro. Nevertheless in response to specific stimulation, we found that monocytes from all three groups responded with a cytokine profile that closely reflected that seen under the same conditions in monocytes from filaria non-endemic normal controls. This observation revealed that monocytes from all filaria-exposed donors principally had the capacity to react to Mf lysate without an inherent defect in one of the patient groups.
Monocytes and macrophages that develop in helminth infections are believed to contribute to wound healing, regulation of Th1 and Th2 inflammation and expulsion of the parasite from the host (reviewed elsewhere [44]). Asymptomatically infected patients are the only filarial-exposed group in which monocytes in the blood come into contact with live microfilariae; thus monocytes may be influenced at this early time point in their differentiation to contribute to immune regulation and therefore the development of asymptomatic infection. Indeed it has been shown that B. malayi microfilariae act on monocytes from filaria non-endemic normal donors to reduce transendothelial migration [45]. To this end, we established that monocytes and macrophages from non-endemic normal donors stimulated with B. malayi Mf lysate in vitro develop a specific phenotype upon activation, and that these cells may influence either the adaptive or innate immune response, respectively.
B. malayi Mf lysate-stimulated monocytes could suppress CD4 + T cell proliferation as well as IFN-c and IL-13 cytokine production in an autologous coculture assay. T cells that received a polyclonal stimulus in the presence of B. malayi Mf lysate-treated monocytes displayed significantly reduced proliferation compared to T cells stimulated in the presence of control monocytes. Furthermore, their ability to produce effector cytokines was significantly inhibited. This is in line with a previous report that demonstrated that PBMCs from microfilaremic patients produce fewer Th1 and Th2 cytokines when stimulated with live microfilariae compared to PBMCs from endemic normals [24].
PD-L1 and IL-10 were significantly induced by Mf lysate stimulation of monocytes and macrophages and thus represented prime candidates responsible for the suppression of T cell responses. Interestingly, neutralisation of IL-10 or PD-1 led to a recovery of CD4 + T cell IFN-c but not IL-13 production. IL-10 has previously been described to be upregulated in adherent cells from patients with lymphatic filariasis [46] and in monocytes from patients harbouring tissue-dwelling filaria [38]. IL-10 has a welldefined role in filarial infections as an immunoregulatory cytokine that regulates both Th1-and Th2-derived inflammatory, potentially harmful responses [47]. PBMCs from asymptomatically infected patients spontaneously secrete significantly higher levels of IL-10 than PBMCs from patients with chronic pathology [46]. Induction of IL-10 is also associated with high levels of immune regulatory IgG4 in human asymptomatic filarial infection (reviewed elsewhere [4]). Thus the high levels of IL-10 observed in our study that resulted from B. malayi Mf lysate stimulation may contribute to T cell suppression in asymptomatic infection.
PD-L1 has been described on monocytes stimulated with live microfilariae in vitro [14]. PD-L1 together with its receptor PD-1 has an important role as a negative costimulator in numerous infection settings [48]. The high mRNA and surface expression of PD-L1 on B. malayi Mf lysate-stimulated monocytes support the idea that microfilaria-modulated monocytes may contribute to asymptomatic infection through this mechanism. Indeed IFN-c responses were also restored after neutralization of PD-1, supporting a role for this molecule.
Neutralisation of IL-10 or PD-1 had only a minimal effect on the recovery of proliferation and had no effect on the restoration of IL-13, implying that other inhibitory mechanisms may be involved. Van der Werf et al. have previously shown that the PD-1-PD-L2 pathway is responsible for Th2 cell hyporesponsiveness in L. sigmodontis infection [49]. Nevertheless this pathway would have been similarly neutralised in our assays through application of the anti-PD-1 antibody, suggesting that other mechanisms play a role in the human T cell impairment observed in our experiments. Other candidates that have been described in murine literature to suppress T cell responses in helminth or other Th2-related diseases through a monocyte/macrophage interaction include arginase [50,51] and RELM-a [52,53], however as mentioned above, these may not represent reliable options in the human system. Further studies describe the roles of retinoic acid or TGF-b in directing the development of Treg cells [54][55][56]; whether the CD4 + T cells in our system have a regulatory phenotype should be further investigated.
In response to stimulation with B. malayi Mf lysate, macrophages upregulated high levels of IL-10, but in contrast to monocytes no significant upregulation of PD-L1 expression was detected. However when B. malayi Mf lysate was added during the differentiation process of macrophages there was a significant and selective impairment in the ability of macrophages to produce cytokines in response to LPS stimulation. Interestingly this was not caused by a reduction in cell survival or a change in cell activation markers. Similar results have been published by Semnani et al. for monocyte-derived dendritic cells stimulated with microfilarial antigen during the differentiation process [57]. Importantly, our results demonstrated that subsequent stimulation with LPS of macrophages differentiated in the presence of Mf lysate resulted in diminished IL-6, TNF-a and IL-12p40 production but not IL-10. This could be a result of distinct signalling pathways being activated in Mf lysate-differentiated macrophages and should be a subject of further studies. The selective impairment of expression of proinflammatory cytokines hints towards a possible involvement of NF-kB1 p50 homodimers, as shown previously [58].
It has been previously demonstrated that B. malayi live microfilariae or live microfilariae in a transwell do not induce phagocytosis in monocytes compared to monocytes exposed to M-CSF for 48 h [14]. The authors of this study argued that microfilariae failed to promote phagocytosis. In contrast we believe that B. malayi microfilariae (or, in our case, B. malayi Mf lysate) directly inhibit phagocytosis of macrophages as a form of immunomodulation. The location of macrophages in the tissues compared to monocytes in the blood may place macrophages in a more advantageous position to phagocytose. In another study, W. bancrofti endemic normal monocytes were incubated with serum from the different groups (endemic normal, patients with lymphatic pathology, microfilaremic patients) [23]. Only monocytes incubated with serum from microfilaremic patients had reduced levels of spreading but not phagocytosis. Thus future studies should determine in detail whether inhibition of phagocytosis actually translates to an increase in microfilarial survival, and which other cells or serum components contribute to microfilarial killing and phagocytosis in vivo.
In conclusion this study has elucidated the monocyte phenotype in patients with active filarial infection and the regulatory capacity of this cell. By directly acting on monocytes in the blood, microfilariae may regulate the antigen-specific T cell response. Furthermore microfilariae may interfere with the differentiation process of macrophages, thus altering their ability to respond to unrelated stimuli in the tissues. The extent to which these findings promote parasite survival and transmission is unclear and should be further investigated.  Figure S3 Monocytes stimulated with excretory/secretory (ES) products from live B. malayi microfilariae do not impair CD4 + T cell proliferation or cytokine production. 5610 5 CFSE-labelled CD4 + T cells were incubated with 1610 5 monocytes left unstimulated (open circles) or stimulated for 24 h with B. malayi microfilarial ES (closed circles) for 3 to 5 days. Proliferation (measured as CFSE dilution) of CD4 + T cells was measured by flow cytometry. Cytokine expression was measured in the culture supernatant by ELISA. Horizontal dashed line indicates the limit of detection of the assay (pooled data from 2 experiments; n = 5-6). All data are represented as mean 6 SEM. P values were calculated using the Wilcoxon signed-rank test. (TIF) Figure S4 Neutralisation of IL-10 in cocultures with unstimulated monocytes. 5610 5 CFSE-labelled CD4 + T cells were incubated for 5 days with 1610 5 unstimulated monocytes +/2 neutralizing antibodies (Ab) for A) IL-10 (pooled data from 4 experiments; n = 12) or B) PD-1 (pooled data from 3 experiments; n = 9). Proliferation (measured as CFSE dilution) of CD4 + T cells was measured by flow cytometry. Cytokine production was measured in the culture supernatant by ELISA. Horizontal dashed line indicates the limit of detection of the assay. All data are represented as mean 6 SEM. P values were calculated using the Wilcoxon signed-rank test. ** p,0.01, *** p,0.001. (TIF) Figure S5 B. malayi Mf lysate-stimulated monocytes express IL-10 mRNA at the time of coculture. Monocytes were stimulated for 4 h or 24 h with B. malayi Mf lysate. mRNA expression was determined using RT-PCR (pooled data from 3 experiments; n = 9). All data are represented as mean 6 SEM. P values were calculated using the Wilcoxon signed-rank test. * p, 0.05. (TIF) Figure S6 Mf lysate but not Fem lysate induces cytokine production in human monocytes. Human monocytes were stimulated for 24 h with 20 mg/ml microfilarial (Mf) or female (Fem) lysate and cytokines were assessed in the supernatant (pooled data from 4-8 experiments; n = 12-24). Data are represented as mean 6 SEM. P values were calculated using the Wilcoxon signed-rank test. ns, not significant. ** p,0.005. (TIF)