6–8 weeks old female BALB/c mice were used for all the experiments in accordance with our Institutional Animal Ethics Committee (IAEC) guidelines. Animals were housed in polypropylene cages (five animals per cage) and kept at our institute’s laboratory animal facility under standard pathogen-free conditions of temperature (24 ± 1°C) and humidity (55–68%) and fed standard pellet diet and water ad libitum. Brugia malayi was maintained in Mastomys coucha and the third infective larval stage of the parasite (Bm-L3, n = 50) recovered from infected Aedes aegypti were used to infect mice via the intra-peritoneal (i.p.) route. Control animals were administered sterile PBS (i.p.).

cDNA synthesis kit, SYBR green master mix, Trizol reagent, DQ-ovalbumin, anti-mouse monoclonal antibodies viz. F4/80, toll-like receptor (TLR)-2, TLR-4, TLR-6, TLR-9, CD69, FITC-labeled secondary IgG antibody, Annexin V Apoptosis Detection Kit, and Caspase sampler assay kit were purchased from Thermo Fischer Scientific (Waltham, MA, USA). Function blocking antibodies viz. anti-CD25 and anti-GITR were purchased from either Thermo Fischer Scientific or BioXcell (West Lebanon, USA). May Grunwald-Giemsa stain was purchased from Merck and Co. (Darmstadt, Germany). CD11c and CD4 magnetic cell separation kit (MACS) were purchased from Miltenyi Biotec (Bergisch-Gladbach, Germany). Fixation and permeabilization kit, Brefeldin A, cell strainer, RBC lysis buffer, anti-mouse monoclonal antibodies viz. CD11c, CD11b, CD80, CD86, MHC-II, TNF-α, IL-4, IL-12, IL-10, Gr-1, Siglec-F, CD4, CD25, FoxP3, CD19, CD5a, and CD1d were purchased from BD Biosciences (San Jose, CA, USA). Anti-mouse monoclonal antibodies Arginase-1 and NOS2 and CCL22 ELISA kit were purchased from R&D biosystems (Minneapolis, MN, USA). NF-κB, p-p38, and p-ERK antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). MEK inhibitor PD0325901, protein tyrosine phosphatase (PTP) inhibitor [bpv (phen)], FITC-dextran, and Arginase activity kit were purchased from Sigma (St. Louis, MO, USA). ELISA kits for Prostaglandins E2 (PGE2) and PGD2 were purchased from Cayman chemicals (Ann Arbor, MI, USA). ELISA kits for LXA4 and LXB4 were purchased from Elabscience (Bethesda, MD, USA). Tyrosine Phosphatase Assay kit was purchased from Promega (Madison, USA).

CD11c positive cells were enriched from the spleens of uninfected and Bm-L3 infected mice at day 3 and day 7 post infection using CD11c magnetic beads as described earlier (6). Thereafter, CD11c positive cell fraction (containing mostly DCs and MΦs) was incubated with CD11b-PE-Cy7 and F4/80-Pacific Blue anti-mouse monoclonal antibodies for 20 min at 4°C and flow cytometric data were acquired on five-decade log-scale dot plots displaying forward scatter (FSC) area vs. side scatter area (SSC). First hierarchy gate was set in FSC-A vs. SSC-A dot plot to exclude contaminating dead cells and cell debris, second hierarchy gate was set in FSC-A vs. FSC-H dot plot to exclude cell doublets and, thereafter, splenic MΦs present within the FSC-A vs. FSC-H dot plot were identified as CD11b^pos, F4/80^pos cells as outlined in Figure 1A. Similarly, eosinophils, inflammatory monocytes, and regulatory B-cells (Bregs) were immunophenotyped from erythrocyte free single cell suspension of spleens based on the differential expression of following markers viz. Siglec F, MHC II, Gr-1, CD11b, CD19, CD1d, and CD5. Briefly, dot plot of FSC-A vs. SSC-A was used to identify granulocytes (low FSC-A, high SSC-A), and further sub-gating of this population helped in the identification of eosinophils (Siglec F^hi, MHC II^neg). Monocytes were identified as Gr-1^hi, CD11b^pos cells, while Bregs were identified as CD19⁺, CD1d^mid, CD5a^hi cells. Data were acquired on FACS Aria flow cytometer and compensation and data analysis was done using FACS DiVA software (BD Biosciences).

Splenic MΦs were identified using the gating strategy as described above. MΦs were sorted using a high-speed FACS Aria flow cytometer fitted with a 70 µm nozzle as described recently (6–8). Sorted MΦs were subjected to post-sort analysis to ascertain the purity of sorted cells and a small fraction was used to prepare cytospins. Remaining cells were used for other immunological assays as detailed below.

CD11c^pos cells (n = 4 × 10⁶) were stained with CD11b-PE-Cy7 and F4/80-Pacific Blue monoclonal antibodies and distributed into four separate tubes followed by addition of FITC-labeled anti-mouse TLR-2, TLR-4, TLR-6, or TLR-9 antibodies. TLR expression on splenic MΦs was ascertained using the gating strategy as described above (Figure 1A). For estimating intracellular cytokines, CD11c^pos cells were first incubated with Brefeldin A (10 µg/ml) for 6 h, then washed and stained with CD11b-PE-Cy7 and F4/80-Pacific blue monoclonal antibodies. Next, they were fixed, permeabilized, and stained with PE-labeled anti-mouse TNF-α, TGF-β, IL-12, IL-4, and IL-10 monoclonal antibodies in separate tubes. Data were acquired on FACS Aria flow cytometer and compensation and data analysis was done using FACS DiVA software (BD Biosciences) (6, 7, 9).

FACS-sorted splenic MΦs (n = 50,000) were incubated with either FITC-dextran (1 mg/ml) to estimate phagocytosis capacity, or with DQ-ovalbumin (0.5 mg/ml) to measure antigen processing capacity for 1 h in a CO₂ incubator at 37°C and increase in FITC fluorescence was measured as described earlier (7). For analysis of maturation and co-stimulation markers, FACS-sorted splenic MΦs were incubated with anti-mouse monoclonal antibodies viz. CD80, CD86, and MHC-II at 4°C for 20 min and, thereafter, acquired on FACS Aria flow cytometer.

FACS-sorted splenic MΦs (n = 5,000) were cocultured with CD4⁺ T cells (n = 2 × 10⁵) that were purified from the spleens of naïve mice using CD4 magnetic beads (Miltenyi Biotec). After 48 h, mitochondrial activity as a measure of T cell proliferation was measured by XTT assay (6).

Lipid intermediates viz. PGE2, PGD2, Lipoxin A4 (LXA4), and B4 (LXB4) present in the culture supernatant of flow-sorted splenic MΦs from control and Bm-L3-infected mice were quantified by ELISA as per instructions of the respective manufacturer.

Bm-L3-infected mice were administered either MEK inhibitor, PD0325901 (5 mg/kg, i.p.) or PTP inhibitor [bpv (phen)] (0.8 mM, i.p.) in separate experiments. In one set of experiments, Bm-L3-infected mice received PD0325901 at day 0 and were sacrificed at day 3 p.i., while in another set they received two doses of PD0325901 at day 0 and day 3 p.i. and were sacrificed at day 7 p.i. Similarly, for PTP inhibition studies, Bm-L3 infected mice received 0.8 mM of [bpv (phen)] daily between day 4 and day 6 p.i. and were sacrificed at day 7 p.i. Bm-L3-infected mice that did not receive any inhibitor served as control animals for inhibition studies.

Total RNA was isolated, quantified, and reverse transcribed from FACS-sorted splenic MΦs and reactions were run on Step One plus thermal cycler (Applied Biosystems) using the SYBR green master mix as described earlier (10). β-actin was used as the reference gene and mean fold-changes were calculated according to the 2^−ΔΔCT method (11). After reactions were over, melting curve analysis was performed to confirm the specificity of amplicons. Primer sequences used for RT-PCR are listed in Table 1.

FACS-sorted splenic MΦs (n = 200,000) were fixed, permeabilized, and stained with primary antibodies against p-p38 and p-ERK. Thereafter, FITC-labeled secondary IgG antibody was added, and after a brief incubation, cells were washed and acquired on a BD FACS Aria flow cytometer (12). For analysis of transcription factor NF-κB, flow-sorted splenic MΦs were lysed in RIPA buffer containing protease inhibitor cocktail and resolved on 10% SDS-PAGE followed by transfer onto nitrocellulose membrane. Next, membranes were blocked with 3% BSA and probed overnight with primary antibody followed by addition of HRP-conjugated secondary antibody and detected using ECL kit.

FACS-sorted splenic MΦs (n = 2 × 10⁶) were lyzed in RIPA buffer containing protease inhibitor cocktail and centrifuged at 10,000×g for 15 min at 4°C. Protein tyrosine phosphatase (PTP) activity was determined in the supernatant according to instructions of the manufacturer (13). Arginase activity was similarly measured in the supernatant by comparing with a urea standard (14). For quantification of caspase activity, p-nitroaniline labeled peptide substrate was used and absorbance of free pNA was quantified using a spectrophotometer (15).

To neutralize the activity of regulatory T cells, Bm-L3-infected mice were administered either respective IgG isotypes or combination of anti-CD25 + anti-GITR function blocking antibodies (100 µg each, i.v.) at day 4 and day 6 post Bm-L3 infection, 24 h later, spleens were excised and immunological assessments were carried out as described in the study.

Apoptosis of Tregs was assessed by Annexin V-FITC and propidium iodide (PI) staining pattern of CD4⁺CD25⁺ T cells (Tregs) present within the lymphocyte gate (low FSC-A, low SSC-A) as per the instructions of the manufacturer.

Figures represent mean ± SD values derived from at least three independent experiments having at least 3–4 animals/group. Statistical analysis was done by either one-way analysis of variance using Bonferroni’s test (Graph Pad Prism 5.0 version 5.01; Graph Pad, San Diego, CA, USA) or two tailed Student’s t-test. p-Value ≤0.05, ≤0.01, and ≤0.001 between different groups or time points were considered significant, highly significant and very highly significant and marked with *, ** and *** respectively.

Splenic MΦs were immunophenotyped and sorted using flow-cytometry on day 3 and day 7 post Bm-L3 infection as described in the Section “Materials and Methods.” Post-sort analysis showed very high purity (>95%) of sorted cells (Figure 1A). Percentages of splenic MΦs-secreting IL-4 increased at day 3 p.i. (p ≤ 0.05), but those secreting IL-12 decreased (p ≤ 0.01) as compared to uninfected controls. This scenario changed rapidly at day 7 p.i. when splenic MΦs secreting IL-4 decreased significantly (p ≤ 0.05) while those secreting IL-12 and IL-10 increased as compared to day 3 p.i. (p ≤ 0.001 for both). Notably, percentages of splenic MΦs secreting TGF-β increased at day 3 p.i. (p ≤ 0.05) but dropped significantly at day 7 p.i. (p ≤ 0.001, Figure 1B).

Real-time RT-PCR analysis revealed 4.5-fold elevated mRNA expression of Arg-1 at day 3 p.i., which increased further to 8.5-fold at day 7 p.i. Quite contrary to this, mRNA expression of NOS2 in splenic MΦs decreased 3.5-fold at day 3 p.i., but moderately increased to 1.8-fold at day 7 p.i. when compared to uninfected controls (Figure 1C). Importantly, transcript levels of other alternative activation markers viz., Ym-1, which is associated with eosinophil chemotaxis increased 18-fold at day 3 p.i. and 10-fold at day 7 p.i. Similarly, Fizz-1, which dampens Th2 inflammation was upregulated 3.6- and 7.5-fold at day 3 and day 7 p.i., respectively (Figure 1C). Also, key markers asociated with regulatory macrophages, i.e., sphingosine kinase-1 and LIGHT were upregulated by 4- and 150-fold, respectively, at day 7 p.i. (Figure 1D), suggesting that Bm-L3-infected splenic MΦs were indeed phenotypically different at these two time points.

To further confirm our findings, we analyzed expression levels of Arg-1 and NOS2 via flow cytometry and the results showed 4.7- and 8-fold upregulated levels of Arg-1 in Bm-L3-infected splenic MΦs at day 3 and day 7 p.i., respectively, which was in contrast to 2.0- and 3.4-fold downregulated levels of NOS2 observed at day 3 and day 7 p.i., respectively (Figure 1E). Interestingly, this observation was corroborated by 3.5-fold (p ≤ 0.05) and five fold (p ≤ 0.01) increased arginase activity in Bm-L3-infected splenic MΦs at day 3 and day 7 p.i., respectively, as compared to uninfected control animals (Figure 1F). Taken together, these results reflected early perturbations in the cytokine secreting potential of splenic MΦs and underlined a tactical shift from a predominantly alternatively activated state (M2 phenotype) of splenic MΦs at day 3 p.i. to a more polarized regulatory phenotype (Mregs) at day 7 p.i. during the first week of Bm-L3 infection.

We assessed the expression of maturation and co-stimulatory markers on the two phenotypes of splenic MΦs, i.e., M2 MΦs and Mregs at day 3 and day 7 p.i., respectively. While the expression of MHCII and CD86 decreased in M2 MΦs, that of CD80 did not change significantly at day 3 p.i. However, expression of these markers was elevated in Mregs at day 7 p.i. (Figure 2A). Furthermore, M2 MΦs exhibited decreased phagocytic capacity (Figure 2B) (p ≤ 0.05), which contrasted with significantly increased antigen processing capacity (p ≤ 0.001) observed in Mregs at day 7 p.i. (Figure 2C).

To further understand the functional consequences of these findings on the proliferation of T cells, FACS-sorted M2 MΦs (at day 3 p.i.) and Mregs (at day 7 p.i.) were cocultured with purified CD4⁺ T-cells obtained from naïve mice. Results showed that M2 MΦs supported the proliferation of CD4⁺ T cells (Figure 2D; p ≤ 0.001) Mregs did not, which underlined the suppressive nature of these cells. Moreover, significantly elevated level of Prostaglandin E2 (PGE2, p ≤ 0.001), but drastically decreased level of Prostaglandin D2 (PGD2, p ≤ 0.01) was observed in Mregs (Figure 2E), but quite surprisingly, no significant change was observed in the level of other small lipid intermediate viz. Lipoxin A4 (LXA4), and Lipoxin B4 (LXB4). These results showed that in addition to IL-10, elevated levels of PGE2 might have contributed toward polarization of M2 MΦs to Mregs during the early phase of Bm-L3 infection.

To gain deeper insights into the molecular pathways that regulated the polarization of splenic MΦs, we assessed the expression of toll-like receptors on MΦs via flow cytometry. Results presented as histograms (Figure 3A) along with mean fluorescence intensity (MFI) values given in Table 2 showed that expression of TLR2, TLR4, TLR6, and TLR9 increased in M2 MΦs and Mregs at day 3 at day 7 p.i. as compared to uninfected controls. However, no change was observed in the transcript level of TLR 5 between M2 MΦs and Mregs (data not shown). Furthermore, while the expression of p-p38 remained unaltered, that of p-ERK was significantly upregulated in M2 MΦs at day 3 p.i. (Figure 3B; p ≤ 0.001; MFI = 6,139 ± 785); however, it was downregulated in Mregs at day 7 p.i. (Figure 3B; p ≤ 0.01, MFI = 3,611 ± 164) when compared to uninfected controls (Figure 3B; MFI = 2,885 ± 243).

Additionally, since the activation of MAPKs is counter-regulated by several PTPs, we estimated PTP activity and found that it was significantly upregulated in Mregs at day 7 p.i. as compared to M2 MΦs (Figure 3C, p ≤ 0.01). Further mRNA analysis revealed that six different PTPs viz., Ptprh, Ptprn, Ptprr, Ptpn21, Ptpn23, and Ptp4a were significantly upregulated in Mregs, which suggested their involvement in inducing hypo-responsive state in Mregs (Figure 3D).

We also analyzed the phosphorylation pattern of two major subunits of NF-κB viz. p65 and p50/105 and found that expression of p65 and p50/105 was downregulated in M2 MΦs, but it was severely impaired in Mregs (Figure 3E). To confirm whether MAPKs regulated this process of polarization, we administered MEK inhibitor PD0325901 to Bm-L3-infected mice and found that ERK inhibition did not affect the development of M2 phenotype, but it significantly altered the polarization of M2 MΦs to Mregs as was evident from higher percentages of IL-4-secreting MΦs (p ≤ 0.05) at D7 post PD0325901 treatment (Figure 3F). Interestingly, administration of PD0325901 did not affect the IL-10 secreting potential of MΦs following treatment. Similarly, to confirm the role of PTPs in the polarization of M2 MΦs to Mregs, we administered PTP inhibitor [bpv (phen)] to Bm-L3-infected mice daily between day 4 and day 6 p.i. and found increased percentages of MΦs-secreting IL-4, but not IL-10 at day 7 p.i. (Figure 3G, p ≤ 0.001). These data suggest that complex mechanisms regulate the process of MΦ polarization and the parasite cleverly exploits these intricately wired molecular pathways to aid its survival.

Based on our previous observation of increased IL-10 secretion by Mregs at day 7 p.i., we reasoned if this would have a bearing on the numbers of Tregs present in the spleens of mice. Indeed, we observed increased percentages of Tregs at day 7 p.i. (4.2 ± 0.91%; Figure 4A) as compared to control animals (2.7 ± 0.14%, Figure 4A). This observation was also complemented by significantly increased concentration of CCL22, a Treg chemoattractant in the culture supernatant of FACS-sorted MΦs at day 7 p.i. (p ≤ 0.01, Figure 4B) and in the peritoneal lavage of Bm-L3-infected mice at day 7 p.i. (p ≤ 0.01, Figure 4B). Together, these results supported the notion that CCL22 driven Treg recruitment might have helped in the polarization M2 MΦs to Mregs. To further confirm our findings that Tregs were indeed driven by elevated CCL22 gradient and were not a result of de novo recruitment from the peripheral blood as might be the case during apoptosis, we checked the percentages of apoptotic T cells present within the CD4⁺CD25⁺ and CD4⁺CD25⁻ gate and quite expectedly found no difference between the control and Bm-L3-infected animals either at day 3 or day 7 p.i. (Figure 4C). To strengthen our reasoning even further, we blocked the activity of Tregs by co-administering neutralizing antibodies against CD25 and GITR (i.v.) at day 4 and day 6 post Bm-L3 infection (Figure 4D) and quite notably found significantly depleted percentages of Tregs (Figure 4E), along with drastically reduced Bm-L3 burden in treated animals (Figure 4F). Not only this, Bm-L3 recovered from anti-CD25 + anti-GITR treated animals were dead and surrounded by thousands of host cells attached to their surface, which showed that anti-CD25 + anti-GITR treatment suppressed the activity of Tregs and boosted anti-parasitic Th2 immunity (Figure 4G).

To evaluate the effect of anti-CD25 + anti-GITR treatment on the polarization of splenic MΦs, we measured IL-4 and IL-10 levels at day 7 p.i., i.e., 24 h after the last dose of anti-CD25 + anti-GITR antibodies and found significantly reduced IL-10 (p ≤ 0.05), but heightened IL-4 secretion (p ≤ 0.05) as compared to mice that received matching IgG isotypes (Figure 5A). Moreover, anti-CD25 + anti-GITR treatment also restored the T cell proliferation capacity of splenic MΦs (p ≤ 0.05, Figure 5B) and increased the expression of T cell activation marker CD69 on CD4⁺ T cells (Figure 5C). Activated CD4⁺ T cells also secreted significantly lower amount of IL-10 (p ≤ 0.05) but more IL-4 (p ≤ 0.05) as compared to IgG controls (Figure 5D). These results further strengthened the notion that anti-CD25 + anti-GITR treatment checked the polarization of M2 MΦs to Mregs and strengthened the antiparasitic immune response in treated animals.

To elucidate whether anti-CD25 + anti-GITR treatment had any effect on the accumulation of leukocytes, we carried out flow cytometry assisted multicolor immunophenotyping of different leukocyte populations present in the spleens of mice. We found increased percentages of granulocytes (low FSC-A, high SSC-A) in the anti-CD25 + anti-GITR-treated mice as compared to IgG controls (Figure 6A). Further confirmation revealed that most of these granulocytes were indeed eosinophils (Figure 6B, SigF⁺, MHCII⁻ cells) that were present almost four times more in anti-CD25 + anti-GITR-treated mice. However, unlike eosinophils, percentages of regulatory B cells (CD1d⁺, CD5a^hi) and inflammatory monocytes (Gr1⁺, CD11b⁺) decreased in anti-CD25 + anti-GITR treated mice as compared to IgG controls (Figures 6C,D). These results underlined the efficacy of anti-CD25 + anti-GITR treatment in breaking immunological tolerance in mice.

Since administration of anti-CD25 + anti-GITR antibodies helped in retaining the M2 phenotype in MΦs, which contributed to reduced Bm-L3 burden, we reasoned if this treatment increased the longevity of M2 MΦs by interfering with their apoptotic machinery. We, therefore, assessed the role of caspases and found reduced expression of caspase 3 (p ≤ 0.001), and caspase 6 (p ≤ 0.01) in the cell lysate of FACS-sorted splenic MΦs from Bm-L3-infected mice that received anti-CD25 + anti-GITR treatment as compared to those that received IgG isotypes (Figure 7). Notably, no change was observed in the activity of caspase 2, 8, and 9, which not only suggested differential regulation of caspases during Bm-L3 infection, but also underlined reduced caspase activity as one of the reasons that might have prolonged survival of M2 MΦs that lead to enhanced killing of infective larvae (16).