IMMUNOLOGY

ORIGINAL ARTICLE

Wolbachia endosymbiont of Brugia malayi elicits a T helper type 17-mediated pro-inflammatory immune response through Wolbachia surface protein Manisha Pathak,1 Meenakshi Verma,1,2 Mrigank Srivastava1,2 and Shailja Misra-Bhattacharya1,2 1

Parasitology Division, CSIR-Central Drug Research Institute, Lucknow, and 2Academy of Scientific and Innovative Research, New Delhi, India

doi:10.1111/imm.12364 Received 2 December 2013; revised 18 July 2014; accepted 21 July 2014. Correspondence: Mrigank Srivastava and Shailja Misra-Bhattacharya, Parasitology Division, CSIR-Central Drug Research Institute, BS 10/1, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow 226031, India. Emails: [email protected]; [email protected] Senior author: Shailja Misra-Bhattacharya

Summary Wolbachia is an endosymbiotic bacterium of the filarial nematode Brugia malayi. The symbiotic relationship between Wolbachia and its filarial host is dependent on interactions between the proteins of both organisms. However, little is known about Wolbachia proteins that are involved in the inflammatory pathology of the host during lymphatic filariasis. In the present study, we cloned, expressed and purified Wolbachia surface protein (r-wsp) from Wolbachia and administered it to mice, either alone or in combination with infective larvae of B. malayi (Bm-L3) and monitored the developing immune response in infected animals. Our results show that spleens and mesenteric lymph nodes of mice immunized with either r-wsp or infected with Bm-L3 show increased percentages of CD4+ T helper type 17 (Th17) cells and Th1 cytokines like interferon-c and interleukin-2 (IL-2) along with decreased percentages of regulatory T cells, Th2 cytokines like IL-4 and IL-10 and transforming growth factor b (TGF-b) levels in culture supernatants of splenocytes. These observations were stronger in mice immunized with r-wsp alone. Interestingly, when mice were first immunized with r-wsp and subsequently infected with Bm-L3, percentages of CD4+ Th17 cells and Th1 cytokines increased even further while that of regulatory T cells, Th2 cytokines and TGF-b levels decreased. These results for the first time show that r-wsp acts synergistically with Bm-L3 in promoting a pro-inflammatory response by increasing Th17 cells and at the same time diminishes host immunological tolerance by decreasing regulatory T cells and TGF-b secretion. Keywords: lymphatic filariasis; T helper type 17; T regulatory cells; Wolbachia; Wolbachia surface protein.

Introduction Lymphatic filariasis caused by Wuchereria bancrofti, Brugia malayi and Brugia timori is the second leading cause of permanent and long-term disability worldwide. Filarial nematodes establish long-term infection through down-modulation of the host immune response leading to long-term debilitating disease. However, for development and survival of lymphatic filariae, an endosymbiotic a-proteobacterium known as Wolbachia is needed. Though Wolbachia were first identified in the ovaries of Culex mosquitoes in 1924 and are known to be concentrated in the intra-cytoplasmic vacuoles of hypodermal lateral cords and female reproductive organs as well as in ª 2014 John Wiley & Sons Ltd, Immunology, 144, 231–244

oocytes, microfilaria thereby reflecting maternal inheritance,1 recent research has indicated their contribution in propagating innate inflammatory responses in human filariasis which has led to a resurgence of interest in these endosymbionts as a means by which to control insecttransmitted diseases.2 Several studies have suggested that elimination of Wolbachia by antibiotic treatment leads to infertility of the female worms, inhibition of larval moulting, and atrophy and death of adult worms.3–5 These findings have prompted the study of Wolbachia as a target for anti-filarial nematode chemotherapy. Also, multiple in vitro and in vivo studies, including clinical trials in humans using Wolbachia-targeting antibiotics have reported anti-filarial effects confirming the essential role 231

M. Pathak et al. that Wolbachia plays in worm survival and hence the vulnerability of elimination.6–10 Wolbachia also mediates a novel form of filarial tolerance to innate inflammatory stimuli, which contributes an additional level of immune cell regulation in the face of chronic filarial infection.11 Recently, a number of regulatory factors, including regulatory T (Treg) cells, interleukin-10 (IL-10), transforming growth factor-b (TGF-b), cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed death 1, have been implicated in establishment of chronic viral, bacterial and parasitic infections.12 After filarial infection, the endosymbionts or their products induce innate inflammatory responses of macrophages13 and neutrophils14 and also activate multiple regulatory responses with separate mechanisms affecting distinct activation signalling pathways11 that skew the host immune response from a moderate pro-inflammatory T helper type 1 (Th1) response to a vigorous Th2-dominated response.15 While the nature of the immune response to parasite antigens in respect of Th1 and Th2 has been characterized in patients and in mouse models, relatively little is known about members of the outer membrane protein family of Wolbachia known as Wolbachia surface proteins (WSP) and their interaction with the filarial host, which is important in maintaining the obligate Wolbachia–Brugia symbiotic relationship, especially in light of recent findings showing that WSP might be secreted by Wolbachia into the worm’s tissue.16 Recently, a recombinant form of the major surface protein of Wolbachia (WSP) has been shown to activate macrophages and dendritic cells via signalling through Toll-like receptor 2 (TLR2) and TLR417 but despite pieces of evidence that highlight their important roles in bacterial pathogenesis, including a function in enhancing the adaptability of bacterial pathogens to various environments,18 the exact function of many of these proteins in development and survival is not yet known. WSP-like proteins are known to correlate with human host immune responses in filarial infection and pathogenesis19 and work by Brattig et al. has provided evidence about a member of WSP family that induces an inflammatory response associated with the pathogenesis of onchocerciasis through the activation of innate immune responses and that possesses anti-apoptotic activity by delaying the apoptosis in human polymorphonuclear cells, which are essential for the initiation and execution of the innate immune response against bacterial pathogens.17,20 Hence Wolbachia and WSP not only activate immune pathways, possibly through TLR which leads to production of reactive oxygen species, proinflammatory cytokines and up-regulation of co-stimulatory molecules that assist in development of inflammation,17,21 but also contribute to the proinflammatory milieu through a biased Th1 response. In the present study we tested the hypothesis that WSP 232

might be one of the major components of Wolbachia that plays an active role in regulating host inflammatory responses as well as inducing immune tolerance in the host upon infection. To accomplish our aim, we cloned, expressed and purified WSP from Wolbachia (r-wsp) and administered it either alone or in combination with infective larvae of B. malayi (Bm-L3) into naive mice and monitored the developing Th1, Th2, Th17 and Treg cell responses during the acute phase of infection. This was primarily done because B. malayi cannot complete its life cycle in immune-competent laboratory mice22–24 but the short-term survival of L3 stage in mice (1–3 weeks) offered us the opportunity to study the early response of WSP in infected animals.

Materials and methods Animals Male BALB/c mice between 6 and 8 weeks of age were used for all experimental studies. Animals were fed a standard pellet diet and water ad libitum under pathogenfree conditions and were housed in our institute’s laboratory animal facility.

Ethics statement All the animals and experimental procedures were duly approved by the Animal Ethics Committee of CDRI, duly constituted under the provisions of the Committee for the Purpose of Control and Supervision on Experiments on Animals, Government of India. The study bears approval no. IAEC/2011/145 dated 03/07/2012.

Reagents and antibodies Freund’s complete adjuvant and Freund’s incomplete adjuvant were purchased from Sigma (St Louis, MO). Monoclonal antibodies and recombinant cytokines for flow cytometry, e.g. phycoerythrin-conjugated IL-2 (IL-2PE; clone-JES6-5H4), IL-4-PE (clone-BVD4-1D11), IL-10-PE (clone-JES5-16E3), interferon-c (IFN-c) -PE (clone-XMG1.2), IL-17-PE (clone-TC11-18H10), FoxP3PE (clone-MF23), CD4-FITC (clone-H129.19), anti-CD28 antibody (clone-37.51), CTLA-4-PE (clone-UC10-4F1011), CD25-PE (clone-PC61), recombinant mouse IL-1b, mouse IL-6, mouse TGF-b, CD3 antibody (clone-1452C11), isotype controls and TGF-b ELISA kit were all purchased from BD Biosciences (San Jose, CA). The magnetic cell separation (MACS) kit and CD4 beads for the isolation of CD4+ T cells from spleens and mesenteric lymph nodes (mLN) were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). Brefeldin A, seroblock FcR, fixation and permeabilization buffer were purchased from BD Biosciences. ª 2014 John Wiley & Sons Ltd, Immunology, 144, 231–244

WSP mediates pro-inflammatory response Cloning, expression and purification of r-wsp and trehalose-6-phosphate phosphatase Wolbachia surface protein from B. malayi was cloned, expressed and purified as described below. Briefly, the wsp gene with NCBI Accession no. AJ252061 was amplified from cDNA of adult B. malayi using forward primer 50 -GAATTCGATCCTGTTGGTCCGATAGCTGATGAGG30 and reverse primer 50 CTCGAGGAGATTAAACGCT ATTCCAGCTTCTGCAC-30 containing restriction sites for EcoRI and XhoI (underlined), respectively and sub-cloned into expression vector pET-28a. The pET28a–Bm-wsp construct was transformed in competent Escherichia coli (DE3) BL21 and culture was induced with 05 mM isopropyl-D thiogalactoside for 5 hr at 37° in logarithmic phase. Culture containing the recombinant protein was solubilized by sonication in solubilization buffer (100 mM NaH2PO4, 100 mM Tris–HCl, pH 80, 200 mM NaCl with 6 M urea) and left overnight at 4° (with shaking) in solubilization buffer. Thereafter, the sample was centrifuged at 13 000 g for 15 min and the supernatant containing the recombinant protein was purified by nickel nitrilotriacetic acid agarose affinity column.25 The purified protein was dialysed overnight against 50 mM NaH2PO4 at 4° and protein concentration was determined using the Bradford method.26 The recombinant protein (r-wsp) showed as a single band of ~25 000 molecular weight on a 10% SDS–PAGE and had endotoxin levels ≤ 1 EU/mg as determined by the E-toxate assay (Sigma). The trehalose 6 phosphate phosphatase gene (tpp) was PCR amplified using cDNA prepared from the adult worms of B. malayi, subcloned into pET28a(+) expression vector and transformed in BL21 E. coli cells. Cells were grown and recombinant trehalose-6-phosphate phosphatase protein (r-tpp) was over-expressed and purified as described earlier.27

Collection of Bm-L3 and infection of mice Bm-L3 were recovered from infected Aedes aegypti that were maintained in the insectarium of our Institute. To elucidate the nature of the immune response in infected animals, we divided mice into five different experimental groups of five or six animals each. Mice in group 1 were given PBS (control group) whereas in group 2 they were given Freund’s complete adjuvant without protein. Mice in group 3 were challenged with 50 live Bm-L3 and in group 4 they were given either 25 lg of r-wsp or r-tpp. Finally, mice in group 5 were first immunized with either 25 lg of r-wsp or r-tpp, followed by infection with 50 live Bm-L3. Administration of PBS, adjuvant, r-wsp and r-tpp was subcutaneous while Bm-L3 were administered intraperitoneally. The experiment was repeated thrice using same number of mice in each group.

ª 2014 John Wiley & Sons Ltd, Immunology, 144, 231–244

Immunization of mice For immunization studies, mice were subcutaneously immunized on day 0 with 25 lg of r-wsp or r-tpp emulsified in Freund’s complete adjuvant. This was followed by two booster doses on weeks 2 and 3 in Freund’s incomplete adjuvant. One week after the final booster dose, mice were infected with 50 live Bm-L3 and killed 1 week later. Spleens and mLN were collected and immunological studies were carried out as described below.

Cell isolations and purification of CD4+ T cells Mice from each group were killed and their spleens and mLN were excised aseptically from their peritoneal cavities. These were subsequently cut into small pieces and gently passed over a 70 lm cell strainer in RPMI-1640 medium to obtain single-cell suspensions. Cells were pelleted by centrifugation at 300 g for 10 min at 4° and red blood cell contaminants in spleen were lysed by a brief exposure to 083% chilled NH4Cl (1 : 3). Erythrocyte-free cells were washed, counted and re-suspended in complete RPMI-1640 medium. Intracellular cytokines were estimated as described earlier.28 In select experiments, to enumerate the percentages of Treg and Th17 cells present in the secondary lymphoid organs of mice, CD4+ T cells were purified from single-cell suspensions using a CD4+ MACS kit from Miltenyi Biotec. Briefly, single-cell suspensions from spleen and mLN were spun at 1200 rpm for 10 min at 4° and the pellet was resuspended in MACS buffer. After a brief centrifugation step, the cells were incubated with seroblock FcR on ice for 10 min to block non-specific antibody binding, then washed with MACS buffer and incubated with CD4 beads (10 ll of beads per 1 9 107 cells) for 15 min at 4°. After incubation, cells were washed, centrifuged and passed over a MACS MS column that was gently flushed with MACS buffer to purify the CD4+ T cells. Aliquots of the resultant cells were subjected to FACS analysis of CD4 cell surface expression and were found to comprise approximately 85–90% of CD4+ cells.

Estimation of intracellular cytokines For estimation of intracellular cytokines, 4 9 106 erythrocyte-free splenocytes and mLN cells were incubated with 10 lg/ml of Brefeldin A (BD Biosciences) in the dark for 6 hr in a CO2 incubator set at 37°. After incubation, cells were washed and incubated with mouse seroblock FcR (BD Biosciences) for 10 min to block non-specific binding of antibodies to CD16 and CD32 markers to curtail the background signal. After FcR blocking, cells were again incubated at 4° in the dark in the presence of FITC conjugated CD4 antibody, after which they were washed with PBS, fixed and permeabilized in the presence of

233

M. Pathak et al. fixation and permeabilization buffer (BD Biosciences) for 15 min. After permeabilization, cells were incubated in separate tubes with PE-labelled anti-mouse monoclonal antibodies directed against IFN-c, IL-2, IL-4 or IL-10. Acquisition was carried out using FACS-DIVA software on a FACS Aria cell sorter (BD Biosciences) or FACS-CELLQUEST software on FACS Calibur (BD Biosciences) and analysis was performed using FLOW JO software (Tree star Inc., Ashland, OR). For estimation of the TGF-b levels present in the culture supernatant of splenocytes from mice of different treated groups, ELISA was carried out using a commercially available ELISA kit (BD Biosciences) following the instructions of the manufacturer.

antibody. After 4 days of incubation, wells were washed three times with PBS and a cocktail of anti-mouse antibodies to CD28 (5 lg/ml), IL-1b (15 ng/ml), IL-6 (5 lg/ ml) and TGF-b (5 ng/ml) in complete RPMI-1640 was added. Fresh medium was added on day 3 and on day 5 cells were harvested and washed once with complete RPMI-1640. Wells were subsequently re-stimulated with 50 ng/ml of PMA and 1 lg/ml of ionomycin in the presence of monensin in complete RPMI-1640 and incubated for 4–5 hr. Unpurified splenocytes and mLN were also stimulated with 50 ng/ml PMA and 1 lg/ml ionomycin in the presence of monensin in complete RPMI-1640 and incubated for 4–5 hr. For detection of Th17 cells, both cell fractions (purified and unpurified) were fixed and permeabilized and anti-mouse IL-17-PE antibody was added. After, 20 min of incubation in the dark at 4°, cells were subsequently acquired and analysed by flow cytometry as described above.

Estimation of Treg and Th17 cells Treg and Th-17 cells were counted in both CD4 purified and unpurified cell fractions from spleens and mLN of mice from differently treated groups. Briefly, cells were first incubated with anti-mouse CD4-FITC antibody for 20 min, followed by their fixation and permeabilization and addition of anti-mouse FoxP3-PE antibody. Acquisition and analysis by flow cytometry was performed as described above. Similarly, Th17 cells were enumerated by estimating the release of IL-17, the key marker for Th17 cells. Briefly, CD4+ purified T cells (3 9 106 cells/ well) were plated onto 48-well tissue culture plates that were pre-coated overnight with 10 lg/ml of anti-CD3

1

(a)

2

1

(b)

2

Isolation of peritoneal exudates cells and real-time RT-PCR analysis of Th1 and Th2 cytokines Peritoneal exudate cells were isolated from mice by lavaging the mouse peritoneum with ice cold Dulbecco’s PBS without calcium and magnesium with the help of a 20G needle attached to a 10 ml syringe. Approximately 8 ml of lavage was collected from each mouse. Cells were then centrifuged at 300 g for 10 min at 4° and supernatant

3

700 bp 650 bp (Amplified product)

500 bp

1500 bp

650 bp (Insert)

500 bp

(c)

(d) 1

2

3

4

1

72 000

(e)

2

2

3

72 000

55 000

72 000

55 000

55 000

36 000

36 000 28 000

36 000

28 000 25 000

28 000

25 000 17 000

17 000 17 000

234

1

25 000

Figure 1. Cloning, over-expression, purification and Western blot analysis of recombinant Wolbachia surface protein (WSP). (a) Amplification of wsp from cDNA of adult female worms by PCR. (b) Restriction map of expression vector pET 28a with 650 bp inserts. (c) Expression optimization of recombinant WSP observed on 10% SDS–PAGE after Coomassie blue staining. (d) Confirmation of expression of WSP recombinant protein by Western blot with anti-His antibody (lane 1 marker, lane 2 recombinant protein). (e) Confirmation of expression of WSP recombinant protein by Western blot with anti-WSP.

ª 2014 John Wiley & Sons Ltd, Immunology, 144, 231–244

M. Pathak et al. fixation and permeabilization buffer (BD Biosciences) for 15 min. After permeabilization, cells were incubated in separate tubes with PE-labelled anti-mouse monoclonal antibodies directed against IFN-c, IL-2, IL-4 or IL-10. Acquisition was carried out using FACS-DIVA software on a FACS Aria cell sorter (BD Biosciences) or FACS-CELLQUEST software on FACS Calibur (BD Biosciences) and analysis was performed using FLOW JO software (Tree star Inc., Ashland, OR). For estimation of the TGF-b levels present in the culture supernatant of splenocytes from mice of different treated groups, ELISA was carried out using a commercially available ELISA kit (BD Biosciences) following the instructions of the manufacturer.

antibody. After 4 days of incubation, wells were washed three times with PBS and a cocktail of anti-mouse antibodies to CD28 (5 lg/ml), IL-1b (15 ng/ml), IL-6 (5 lg/ ml) and TGF-b (5 ng/ml) in complete RPMI-1640 was added. Fresh medium was added on day 3 and on day 5 cells were harvested and washed once with complete RPMI-1640. Wells were subsequently re-stimulated with 50 ng/ml of PMA and 1 lg/ml of ionomycin in the presence of monensin in complete RPMI-1640 and incubated for 4–5 hr. Unpurified splenocytes and mLN were also stimulated with 50 ng/ml PMA and 1 lg/ml ionomycin in the presence of monensin in complete RPMI-1640 and incubated for 4–5 hr. For detection of Th17 cells, both cell fractions (purified and unpurified) were fixed and permeabilized and anti-mouse IL-17-PE antibody was added. After, 20 min of incubation in the dark at 4°, cells were subsequently acquired and analysed by flow cytometry as described above.

Estimation of Treg and Th17 cells Treg and Th-17 cells were counted in both CD4 purified and unpurified cell fractions from spleens and mLN of mice from differently treated groups. Briefly, cells were first incubated with anti-mouse CD4-FITC antibody for 20 min, followed by their fixation and permeabilization and addition of anti-mouse FoxP3-PE antibody. Acquisition and analysis by flow cytometry was performed as described above. Similarly, Th17 cells were enumerated by estimating the release of IL-17, the key marker for Th17 cells. Briefly, CD4+ purified T cells (3 9 106 cells/ well) were plated onto 48-well tissue culture plates that were pre-coated overnight with 10 lg/ml of anti-CD3

1

(a)

2

1

(b)

2

Isolation of peritoneal exudates cells and real-time RT-PCR analysis of Th1 and Th2 cytokines Peritoneal exudate cells were isolated from mice by lavaging the mouse peritoneum with ice cold Dulbecco’s PBS without calcium and magnesium with the help of a 20G needle attached to a 10 ml syringe. Approximately 8 ml of lavage was collected from each mouse. Cells were then centrifuged at 300 g for 10 min at 4° and supernatant

3

700 bp 650 bp (Amplified product)

500 bp

1500 bp

650 bp (Insert)

500 bp

(c)

(d) 1

2

3

4

1

72 000

(e)

2

2

3

72 000

55 000

72 000

55 000

55 000

36 000

36 000 28 000

36 000

28 000 25 000

28 000

25 000 17 000

17 000 17 000

234

1

25 000

Figure 1. Cloning, over-expression, purification and Western blot analysis of recombinant Wolbachia surface protein (WSP). (a) Amplification of wsp from cDNA of adult female worms by PCR. (b) Restriction map of expression vector pET 28a with 650 bp inserts. (c) Expression optimization of recombinant WSP observed on 10% SDS–PAGE after Coomassie blue staining. (d) Confirmation of expression of WSP recombinant protein by Western blot with anti-His antibody (lane 1 marker, lane 2 recombinant protein). (e) Confirmation of expression of WSP recombinant protein by Western blot with anti-WSP.

ª 2014 John Wiley & Sons Ltd, Immunology, 144, 231–244

M. Pathak et al. Spleen (a)

104

mLN

Q1 1·30%

0·74%

103

Control

(h)

Q2

101 Q3 34·9%

100 100 101

Isotype

100 101 (i) 104

Q2

0·87%

10

100 101

FMO

0

(j) 104

0·09%

102

10

3

104

10

Q2

0·07%

and r-wsp reacted with anti-wsp antibody giving a characteristic band at ~25 000 including His-tag at C and N termini. Normal serum, used as a negative control, did not bind with r-wsp (Fig 1e).

2

10

101 Q3 30·8% 3 4

100

Adjuvant

2

Q1 0·084%

103

101

IL-2

1

10 10

Q2

100

101

Q1 1·63%

103

10

2

10

100

(k) 104

0·89%

102

101

101 Q3 41·7%

100

101

Q1 1·19%

103

1·06%

104

Administration of r-wsp increases production of proinflammatory cytokines

0·17%

Q3 38·2%

100

(l) 104

103 Q2

Q1 0·317%

100

104 Q2

102

101

Q1 1·27%

103

102

104

103 Q2

0·23%

L3

103

102

10

1

103

102

100

Q3 30·9%

100

10

Q2

102

102

101

101

0

10

100 101 (f) 104

Q1 1·20%

r-wsp

103

102

Q3 45·4% 3 10 104 Q2

Q1 1·72%

102

103 104 Q2

0·33%

102 101

101 Q3 41·9%

100 100

101

10

2

Q1 1·75%

103 Q2

Q3 22·6%

100 100 101

104 (n) 104

2·13%

103

r-wsp + L3

100 101 103

102

(g) 104

Q3 17·5%

100

(m) 104

1·36%

102 103

104

Q2

Q1 2·12%

0·49%

103

102

102

101

101

100 100 101

102

Q3 40·5% 103 104

100 100

101

102

Q3 22·3% 3 10 104

CD4

digestion of the recombinant construct of pET28a-wsp (Fig. 1b). r-wsp had a molecular weight of ~ 25 000 with (His)6-tag fused at the N and C termini in BL-21 E. coli cells (Fig. 1c). The presence of r-wsp was confirmed by an anti-His antibody in the Western blot (Fig. 1d). Polyclonal antibodies were subsequently raised against r-wsp 236

Figure 3. Estimation of interleukin-2 (IL-2) in secondary lymphoid organs of mice. IL-2 was estimated by flow cytometry as described in Materials and methods. Representative FACS dot blots from spleen (a–g) and mesenteric lymph nodes (mLN) (h–n) are shown. Significant differences were observed in spleen between infective larvae of B. malayi (Bm-L3), recombinant Wolbachia surface protein (r-wsp) and Bm-L3+r-wsp treated mice in comparison with control. P ≤ 001, P ≤ 001 and P ≤ 0001 respectively. Similarly, significant differences were reported in mLN between Bm-L3, r-wsp and BmL3+r-wsp treated mice. P ≤ 0001, P ≤ 0001 and P ≤ 0001, respectively. The dot plots are representative of three independent experiments performed on different days with five or six animals per group.

Q3 43·4%

100

102 103 104

Q1 0·098%

103

(e) 104

0·054%

101 Q3 43·9%

100

104

103 104

102

101

(d)

102

Q2

Q1 0·146%

103

102

104

Q3 20·4%

100

102 103 104

Q1 0·190%

3

(c)

0·13%

102

101

104

Q2

Q1 0·591%

103

102

(b)

104

Spleens of mice infected with live Bm-L3 show up-regulation of Th1 cytokines IFN-c (Fig. 2e) (P ≤ 001) and IL-2 (Fig. 3e) (P ≤ 001) compared with uninfected controls (Figs 2a–d and 3a–d). Up-regulation of pro-inflammatory cytokines was marked by significantly down-regulated levels of Th2 cytokines IL-4 (Fig. 4e) (P ≤ 001) and IL-10 (Fig. 5e) (P ≤ 005) as compared with uninfected controls (Figs 4a–d and 5a–d). Similarly, spleens of mice given 25 lg of r-wsp responded with elevated levels of IFN-c (Fig. 2f) (P ≤ 0001) and IL-2 (Fig. 3f) (P ≤ 001) and decreased levels of IL-4 (Fig. 4f) and IL-10 (Fig. 5f) (P ≤ 0001) compared with uninfected controls (Figs 2–5 a–d). Interestingly, spleens of mice immunized with r-wsp and challenged with live Bm-L3 also showed increased levels of IFN-c (Fig. 2g) (P ≤ 0001) and IL-2 (Fig. 3g) (P ≤ 0001) and decreased levels of IL-4 (Fig. 4g) (P ≤ 0001) and IL-10 (Fig. 5g) (P ≤ 0001). Similar results were obtained from analysis of mLN where mice infected with live Bm-L3 showed up-regulation of IFN-c (Fig. 2l) and IL-2 (Fig. 3l) (P ≤ 0001) and down-regulated levels of IL-4 (Fig. 4l) (P ≤ 001) and IL-10 (Fig. 5l) compared with controls (Figs 2–5h–k). Similarly, mLN of mice challenged with 25 lg of r-wsp responded with elevated levels of IFNc (Fig. 2m) and IL-2 (Fig. 3m) (P ≤ 0001) and decreased levels of IL-4 (Fig. 4m) (P ≤ 0001) and IL-10 (Fig. 5m) (P ≤ 0001). Interestingly, mLN of mice immunized with r-wsp and challenged with live Bm-L3 also showed increased levels of IFN-c (Fig. 2n) (P ≤ 0001) and IL-2 (Fig. 3n) (P ≤ 0001) and decreased levels of IL-4 (Fig. 4n) (P ≤ 0001) and IL-10 (Fig. 5n) (P ≤ 0001) compared with uninfected controls (Figs 2–5h–k) mice. These results show that administration of r-wsp leads to an increase in ª 2014 John Wiley & Sons Ltd, Immunology, 144, 231–244

WSP mediates pro-inflammatory response Spleen (a) 104

mLN

Q1 0·642%

105

0·32%

103

Control

(h)

Q2

100 101 Q1 0·046%

103

Isotype

102 0

Q3 39·1%

100 102

(i) 105

Q2

0·01%

FMO

102 0

Q3 43·8%

100 101 Q1 0·091%

102

103 104 105

0·04%

Adjuvant

IL-4

Q1 1·58%

103

102

Q2

(k) 105

0·39%

Q3 22·4%

100 101 Q1 0·454%

102

103 104 Q2

L3

Q1 3·84%

104

105 Q2

0·26%

102

103

Q1 11·7%

104

105 Q2

3·02%

104 103 102 0

Q3 43·2%

100

101

102

103

Q1 0·638%

104 Q2

Q3 19·3%

0 10 (m) 105

0·28%

2

103

Q1 11·4%

104

105 Q2

2·69%

104

102

103

101

102 0

Q3 41·7%

100 100

101

Q1 0·591%

103

102

103

104

Q2

Q3 18·5%

0 (n) 105

0·13%

102

Q1 9·88%

103

104

105 Q2

2·56%

104

102

103

101 Q3 20·4%

0

100 101

102

102 0

103 104

IL-4, IL-10 and TGF-b were also quantified using qRTPCR which exhibited similar pattern (see Supporting information, Fig. S1). Primer sequences are given in Table S1 (see Supporting information).

Administration of r-wsp induces proliferation of Th17 cells

3·14%

Q3 28·8%

0 (l) 105

102

103

r-wsp

103

102 0

101

r-wsp + L3

102

103

103

10

0·51%

104

101

(g) 104

105 Q6

Q7 19·8%

0

103 104

102

104

Q5 0·679%

104

102 0

Q3 26·9%

100 101

(f)

103

103

100

100

102

104

101

Figure 4. Estimation of interleukin-4 (IL-4) in secondary lymphoid organs of mice. IL-4 was estimated by flow cytometry as described in Materials and methods. Representative FACS dot blots from spleen (a–g) and mesenteric lymph nodes (mLN) (h–n) are shown. Significant differences were observed in spleen between infective larvae of B. malayi (Bm-L3), recombinant Wolbachia surface protein (r-wsp) and Bm-L3+r-wsp treated mice in comparison with control. P ≤ 001 and P ≤ 0001, respectively. Similarly, significant differences were reported in mLN between Bm-L3, r-wsp and Bm-L3+r-wsp treated mice. P ≤ 001, P ≤ 0001 and P ≤ 0001, respectively. The dot plots are representative of three independent experiments performed on different days with five or six animals per group.

Q3 39·7%

0 (j)

Q2

102

(e) 104

0·36%

103

103

100

Q2

Q1 0·271%

104

101

(d) 104

Q3 29·9%

0 102 103 104 105

103 104

102

(c) 104

3·81%

103

101

100

Q2

104

102

(b) 104

Q1 4·83%

Q3 20·1%

0 102 103 104 105 CD4

the levels of Th1 cytokines at the cost of Th2 cytokines and that it acts in a synergistic manner with Bm-L3 to promote a biased Th1 response in infected animals. To analyse the changes described above at mRNA level, select Th1 and Th2 cytokines, e.g. IL-2, IFN-c, tumour necrosis factor-a, ª 2014 John Wiley & Sons Ltd, Immunology, 144, 231–244

Interleukin-17 predominantly expressed by Th17 cells has been suggested to play an important role during the chronic stage of filarial infection.31 To study the role of Th17 cells and their interplay with Treg cells, mice were immunized with r-wsp, as described in the Materials and methods. The results show that spleens of mice infected with live Bm-L3 had increased percentages of CD4+ Th17 cells (Fig. 6e) compared with uninfected controls (Fig. 6a–d). However, this increase was more pronounced in spleens of mice that were given r-wsp (P ≤ 005, Fig. 6f) and increased even further in mice that were first immunized with r-wsp and then infected with Bm-L3 (P ≤ 0001, Fig. 6g). Likewise, mLN of infected mice showed increased percentages of CD4+ Th17 cells which gradually increased after introduction of live Bm-L3 (Fig. 6l). The increase in Th17 cell numbers was more pronounced in r-wsp-immunized mice (P ≤ 001, Fig. 6m) and was enhanced even further upon L3 challenge (P ≤ 0001, Fig. 6n) compared with controls (Fig. 6h–k). Similar results were obtained using the CD4 T-cell-purified fraction (see Supporting information, Fig. S2). To confirm that synergism seen between r-wsp and Bm-L3 was responsible for elevating Th17 levels and was not a generalized phenomenon with other parasite proteins, another parasite protein, namely r-tpp, was taken and mice were immunized in the same way as described in the Materials and methods. Our results show that r-tpp when administered either alone or in combination with Bm-L3 failed to increase the percentages of Th17 cells – highlighting that increase in proinflammatory Th17 cells was exclusive to r-wsp (see Supporting information, Fig. S3). 237

M. Pathak et al. Spleen (a)

104

mLN

Q1 1·30%

0·74%

103

Control

(h)

Q2

101 Q3 34·9%

100 100 101

Isotype

100 101 (i) 104

Q2

0·87%

10

100 101

FMO

0

(j) 104

0·09%

102

10

3

104

10

Q2

0·07%

and r-wsp reacted with anti-wsp antibody giving a characteristic band at ~25 000 including His-tag at C and N termini. Normal serum, used as a negative control, did not bind with r-wsp (Fig 1e).

2

10

101 Q3 30·8% 3 4

100

Adjuvant

2

Q1 0·084%

103

101

IL-2

1

10 10

Q2

100

101

Q1 1·63%

103

10

2

10

100

(k) 104

0·89%

102

101

101 Q3 41·7%

100

101

Q1 1·19%

103

1·06%

104

Administration of r-wsp increases production of proinflammatory cytokines

0·17%

Q3 38·2%

100

(l) 104

103 Q2

Q1 0·317%

100

104 Q2

102

101

Q1 1·27%

103

102

104

103 Q2

0·23%

L3

103

102

10

1

103

102

100

Q3 30·9%

100

10

Q2

102

102

101

101

0

10

100 101 (f) 104

Q1 1·20%

r-wsp

103

102

Q3 45·4% 3 10 104 Q2

Q1 1·72%

102

103 104 Q2

0·33%

102 101

101 Q3 41·9%

100 100

101

10

2

Q1 1·75%

103 Q2

Q3 22·6%

100 100 101

104 (n) 104

2·13%

103

r-wsp + L3

100 101 103

102

(g) 104

Q3 17·5%

100

(m) 104

1·36%

102 103

104

Q2

Q1 2·12%

0·49%

103

102

102

101

101

100 100 101

102

Q3 40·5% 103 104

100 100

101

102

Q3 22·3% 3 10 104

CD4

digestion of the recombinant construct of pET28a-wsp (Fig. 1b). r-wsp had a molecular weight of ~ 25 000 with (His)6-tag fused at the N and C termini in BL-21 E. coli cells (Fig. 1c). The presence of r-wsp was confirmed by an anti-His antibody in the Western blot (Fig. 1d). Polyclonal antibodies were subsequently raised against r-wsp 236

Figure 3. Estimation of interleukin-2 (IL-2) in secondary lymphoid organs of mice. IL-2 was estimated by flow cytometry as described in Materials and methods. Representative FACS dot blots from spleen (a–g) and mesenteric lymph nodes (mLN) (h–n) are shown. Significant differences were observed in spleen between infective larvae of B. malayi (Bm-L3), recombinant Wolbachia surface protein (r-wsp) and Bm-L3+r-wsp treated mice in comparison with control. P ≤ 001, P ≤ 001 and P ≤ 0001 respectively. Similarly, significant differences were reported in mLN between Bm-L3, r-wsp and BmL3+r-wsp treated mice. P ≤ 0001, P ≤ 0001 and P ≤ 0001, respectively. The dot plots are representative of three independent experiments performed on different days with five or six animals per group.

Q3 43·4%

100

102 103 104

Q1 0·098%

103

(e) 104

0·054%

101 Q3 43·9%

100

104

103 104

102

101

(d)

102

Q2

Q1 0·146%

103

102

104

Q3 20·4%

100

102 103 104

Q1 0·190%

3

(c)

0·13%

102

101

104

Q2

Q1 0·591%

103

102

(b)

104

Spleens of mice infected with live Bm-L3 show up-regulation of Th1 cytokines IFN-c (Fig. 2e) (P ≤ 001) and IL-2 (Fig. 3e) (P ≤ 001) compared with uninfected controls (Figs 2a–d and 3a–d). Up-regulation of pro-inflammatory cytokines was marked by significantly down-regulated levels of Th2 cytokines IL-4 (Fig. 4e) (P ≤ 001) and IL-10 (Fig. 5e) (P ≤ 005) as compared with uninfected controls (Figs 4a–d and 5a–d). Similarly, spleens of mice given 25 lg of r-wsp responded with elevated levels of IFN-c (Fig. 2f) (P ≤ 0001) and IL-2 (Fig. 3f) (P ≤ 001) and decreased levels of IL-4 (Fig. 4f) and IL-10 (Fig. 5f) (P ≤ 0001) compared with uninfected controls (Figs 2–5 a–d). Interestingly, spleens of mice immunized with r-wsp and challenged with live Bm-L3 also showed increased levels of IFN-c (Fig. 2g) (P ≤ 0001) and IL-2 (Fig. 3g) (P ≤ 0001) and decreased levels of IL-4 (Fig. 4g) (P ≤ 0001) and IL-10 (Fig. 5g) (P ≤ 0001). Similar results were obtained from analysis of mLN where mice infected with live Bm-L3 showed up-regulation of IFN-c (Fig. 2l) and IL-2 (Fig. 3l) (P ≤ 0001) and down-regulated levels of IL-4 (Fig. 4l) (P ≤ 001) and IL-10 (Fig. 5l) compared with controls (Figs 2–5h–k). Similarly, mLN of mice challenged with 25 lg of r-wsp responded with elevated levels of IFNc (Fig. 2m) and IL-2 (Fig. 3m) (P ≤ 0001) and decreased levels of IL-4 (Fig. 4m) (P ≤ 0001) and IL-10 (Fig. 5m) (P ≤ 0001). Interestingly, mLN of mice immunized with r-wsp and challenged with live Bm-L3 also showed increased levels of IFN-c (Fig. 2n) (P ≤ 0001) and IL-2 (Fig. 3n) (P ≤ 0001) and decreased levels of IL-4 (Fig. 4n) (P ≤ 0001) and IL-10 (Fig. 5n) (P ≤ 0001) compared with uninfected controls (Figs 2–5h–k) mice. These results show that administration of r-wsp leads to an increase in ª 2014 John Wiley & Sons Ltd, Immunology, 144, 231–244

WSP mediates pro-inflammatory response Spleen

Control

(a) 105

Q1 0·944%

mLN 0·134%

104

104

103

103

102 0 0 102 103 10

Isotype

(b) 105

Q1 1·62%

105 104

103

103

0 102 103 10

FMO

(i)

104

105 Q1 0·00%

(j)

104

103

103

Adjuvant

IL-17

105

Q1 0·830%

105

0·26%

104

104

103

103

L3

Q1 1·12%

Q2

104

104

103

103

0 102 103 10

r-wsp

(f)

105

Q1 2·54%

Q3 38·0% 4 5

(m) 5 10

0·51%

104

103

103

r-wsp + L3

105

Q1 1·80%

0·78%

104

103

103 102 0

Q3 29·1%

Q2

0·38%

Q1 1·81%

Q3 31·5% 4 5

10

Q2

0·49%

Q3 33·2%

0 102 103 104 105

(n) 105

Q2

104

102 0

Q2

0·28%

Q3 16·9%

102 0

Q3 31·4%

Q1 1·90%

Q1 2·38%

0 102 103 10

10

Q2

0 102 103 104 105

(g)

Q3 27·8%

102 0

104

102 0

0·12%

0 102 103 104 105

(l) 105

0·37%

102 0

Q2

102 0

Q3 37·6%

0 102 103 104 105

(e) 105

Q3 19·1%

0 102 103 104 105

(k)

Q2

102 0

Q2

0·21%

102 0

Q3 27·8%

0 102 103 104 105

(d)

Q1 1·62%

Q1 105 0·00%

104

102 0

Q3 25·0%

0 102 103 104 105

10

0·13%

0·233%

102 0

Q3 19·1% 4 5 Q2

Q2

0 102 103 104 105

10

Q2

Q1 1·66%

102 0

Q3 28·9% 4 5

0·22%

102 0

(c)

(h) 105

Q2

0 102 103 104 105

Q1 6·17%

Q2

0·94%

Q3 15·2%

0 102 103 104 105

CD4

to anti-filarial drug treatment.5 However, it is not clear what role Wolbachia (and derived molecules) may play in driving the acquired immune responses associated with filarial disease pathogenesis. Since the death and disintegration of worms provoke inflammatory reactions, the possibility that other Wolbachia antigens play a role in promoting the immunopathology of filariasis requires ª 2014 John Wiley & Sons Ltd, Immunology, 144, 231–244

Figure 6. Estimation of interleukin-2 (IL-17) in secondary lymphoid organs of mice. IL-17 was estimated by flow cytometry as described in Materials and methods. Representative FACS dot blots from spleen (a–g) and mesenteric lymph nodes (mLN) (h–n) are shown. Significant differences were observed in spleen between infective larvae of B. malayi (Bm-L3), recombinant Wolbachia surface protein (r-wsp) and Bm-L3+r-wsp treated mice in comparison with control. P ≤ 005 and P ≤ 0001, respectively. Similarly, significant differences were reported in mLN between Bm-L3, r-wsp and Bm-L3+r-wsp treated mice. P ≤ 001 and P ≤ 0001, respectively. The dot plots are representative of three independent experiments performed on different days with five or six animals per group.

in-depth investigation.33,34 Previous studies have shown that major surface proteins of Wolbachia (WSP) are highly conserved in Wolbachia and have a heterogeneous pattern of amino acid diversity characteristic of other outer membrane proteins.35–38 Surface proteins of endosymbionts such as WSP are involved in the Wolbachia– Brugia symbiotic interaction,16,19 and previous reports have suggested that the inflammatory potential of filariae is also a result of the presence of Wolbachia,11 which has been implicated in propagating innate inflammatory responses in human filariasis.2 As a result, we investigated the role of WSP in more detail. Our hypothesis was based on reports wherein it has been shown that extracts of B. malayi female worms mediate a complete homotolerant state in murine peritoneal macrophages in terms of pro-inflammatory cytokine production toward a secondary stimulus and a degree of heterotolerance toward multiple TLR ligands. Additionally, it has been shown that B. malayi female worms can also mediate tolerance to a TLR-independent stimulus, CD40 ligand. The ability of B. malayi female worms to render peritoneal macrophages tolerant to TLR and CD40-specific ligands was shown to be dependent on the quantity of Wolbachia contained within parasite tissues and so available as components of the soluble preparation that would ultimately control the response of innate immune cells as well as different T-cell subsets. The role of different T-cell subsets is important during the acute phase of filarial infection because different T-cell subsets inhibit the proliferation of the other subtype, e.g. Th17 cells produce cytokines such as IL-17A, IL-17F, IL-6, tumour necrosis factor-a and IL-21, all of which are associated with inflammation and play active roles in cell-mediated autoimmune diseases, but these cells are tightly regulated by Th1 and Th2 cytokines and FoxP3+ Treg cells. The Treg cells are recruited de novo by the L3 stage of the filarial parasite during initiation of the infection to suppress inflammation.39 Therefore in the present study, we cloned, expressed and purified WSP from Wolbachia and investigated its role in mediating the host T-cell response upon infection. Our results show that secondary lymphoid organs of mice after administration of r-wsp accumulated increased percentages of CD4+ 239

M. Pathak et al. Spleen

Control

(a) 105

mLN Q2

Q1 1·08%

(h)

1·91%

104

104

103

103

102 0 10 (b) 105

2

103

Q1 0·080%

104

Q1 105 0·048%

FMO

Q2

(j)

0·04%

103

103

102

Adjuvant

FoxP3

Q2

104

104

103

103

0

L3

102

Q1 3·14%

103

2·72%

104 103

0

102

Q1 1·71%

103

104

104

103

103

0

r-wsp + L3

Q2

0·94%

103

103 Q3 33·8%

0 102 103 104 105

Q1 1·50%

3

10

10

4

105 Q2

1·15%

Q3 20·8%

0 102 103 104 105 (n) 105 104

0

2

0

104

102

Q2

4·97%

102

0 102 103 104 105 Q1 1·10%

105

Q3 14·5%

0 10 (m) 105

1·02%

Q3 24·8%

104

0

104

102

103

102

105 Q2

102

Q1 105 4·65%

103 Q3 29·7%

Q2

2·90%

Q3 26·0%

0 (l)

104

0

Q3 24·5%

0

105 Q2

102

r-wsp

104

0·04%

Q1 1·79%

102 0

Q1 0·545%

Q2

0·44%

Q3 33·8%

0 102 103 104 105

Th17 cells, which was higher than Bm-L3 infection alone. Moreover, the levels of Th1 cytokines IFN-c and IL-2 were also prominently elevated in r-wsp-immunized mice than Bm-L3-infected animals. These results show that purified r-wsp possesses pro-inflammatory potential that helps in shifting the host immune response towards a Th1 phenotype. This observation is important because 240

Figure 7. Estimation of regulatory T (Treg) cells in secondary lymphoid organs of mice. FoxP3 was estimated by flow cytometry as described in Materials and methods. Representative FACS dot blots from spleen (a–g) and mesenteric lymph nodes (mLN) (h–n) are shown. Significant differences were observed in spleen between infective larvae of B. malayi (Bm-L3), recombinant Wolbachia surface protein (r-wsp) and Bm-L3+r-wsp treated mice in comparison with control. P ≤ 001, P ≤ 001 and P ≤ 001, respectively. Similarly, significant differences were reported in mLN between Bm-L3, r-wsp and Bm-L3+r-wsp treated mice. P ≤ 001, P ≤ 005 and P ≤ 005, respectively. The dot plots are representative of three independent experiments performed on different days with five or six animals per group.

105 Q2

102

Q3 37·4%

0

104

0 102 103 104 105 (k) 105

1·83%

102

103

0

0 102 103 104 105 Q1 1·66%

102

102

Q3 24·5%

0

Q3 42·0%

0

104

(g) 105

0·02%

Q1 105 0·00%

4

(f) 105

Q2

0

0 102 103 104 105 105 Q1 0·00%

(e) 105

105

104

102 Q3 40·5%

0

(d) 105

103

103

102

10

102

104

103

(c)

2·92%

Q3 30·8%

0 (i)

0·04%

104

Q2

0

105 Q2

Q1 2·83%

102

Q3 43·0%

0

Isotype

105

the early human immune response to L3 of B. malayi has not been well-characterized in detail in vivo (because of the inability to determine the precise time of infection) and only in vitro data are available, which have been obtained by culturing human peripheral blood mononuclear cells.15 Our data therefore, reinforce the belief that as in humans, the primary immune response in mice to the live infective stage of the parasite is not predominantly Th2 in nature but rather dominated by a proinflammatory response.15 We also noted decreased percentages of Treg cells and Th2 cytokines IL-4 and IL-10 in secondary lymphoid organs of mice infected with both Bm-L3 and r-wsp. Again, mice receiving r-wsp exhibited more prominent reduction compared with Bm-L3-infected animals. The secreted levels of TGF-b in culture supernatants of splenocytes from mice of different treated groups also support down-regulation of Treg cells. These results are in agreement with previous reports showing that Wolbachia contributes towards a dysregulated and tolerized immunological phenotype that accompanies the majority of human filarial infections.11 It is now well-known that a complex system of regulatory events occurs following activation of immune cells, which among others includes the induction of suppressive cytokines as observed in the present study.40 Interestingly, active B. malayi infection is notable for its ability to induce Th241 polarized immune responses, whereas its endosymbiont Wolbachia induces a Th1-biased immune response21 and both Wolbachia and Wolbachia-derived molecules are known for their pro-inflammatory reactions.42 Interestingly, mice immunized with r-wsp and subsequently infected with Bm-L3 exhibited even further elevated Th1 and Th17 responses and an even more diminished Th2 and Treg response than either treatment alone, suggesting synergistic action of r-wsp and Bm-L3 and reinforcing the belief that Treg cells enjoy an inverse relationship with Th17 cells. Notably, challenging immunized mice with Bm-L3 sheds invaluable light on how the mammalian immune response reacts to, and may be manipulated by, WSP during filarial infection, especially because the L3 stage of the filarial parasite inhibits proª 2014 John Wiley & Sons Ltd, Immunology, 144, 231–244

WSP mediates pro-inflammatory response Spleen

Control

(a)

2·27%

Q5 105 0·709%

104

104

103

103

102 0 2

0 10

(b) 105

103

(i) 105

103

102

102

Q3 42·8%

0 10 10

FMO

104

104

103

103

102 0

Q3 38·0%

102

2·10%

102 0 2

0 10

L3

103

Q5 5·69%

1·89% 104

103

103

r-wsp

1·41%

104

103

103 Q7 15·5%

0

r-wsp + L3

(g) 105

102

Q5 4·75%

103

104

1·26% 104

103

103 Q7 19·7%

Q7 31·7%

0

(n) 105

104

102 0

105

5·73%

Q6

3·84%

102 0

105 Q6

104

Q6

Q5 0·496%

Q5 105 0·499%

104

102 0

103

0 102 103 104 105

(m)

Q6

Q5 5·65%

Q7 32·6%

102

102 0

Q7 20·5%

0 102 103 104 105 10

Q6 0·21%

Q7 20·2%

0

(l) 105

Q6

104

5

105

10

102 0

Q7 22·1% 4 10 105

102 0

(f)

10

Q5 0·121%

4

103

103

(e) 105

3

0 102 103 104 105 Q6 (k) 105 Q5 0·491% 3·14% 104

Q6

104

Adjuvant

CD25

Q5 6·28%

Q7 33·3% 105

0·57%

102 0

Q7 20·4%

0 102 103 104 105

(d) 105

10

0

(j) 105

Q6 0·25%

4

Q2

Q1 0·452%

0 Q5 0·169%

3

104

103

(c) 105

5·21%

2

0·78%

0

Q6

102 0

Q7 18·0% 4 10 105 Q2

Q1 0·613%

104

Isotype

mLN

(h)

Q6

105 Q5 7·52%

102 0

0 102 103 104 105

102

Q5 0·254%

103

104

Q7 36·5% 105 Q6

2·82%

Q7 32·2%

0 102 103 104 105 CD4

tective immunity and increases survival of parasites by increasing expression of Treg cells.43 In susceptible mice, the Th1 response is known to facilitate L3 development while resistant mice that do not support L3 development lack the pre-patent Th1 response.15,44 Our study shows the development of a ª 2014 John Wiley & Sons Ltd, Immunology, 144, 231–244

Figure 8. Estimation of CD25 in secondary lymphoid organs of mice. CD25 was estimated by flow cytometry as described in Materials and methods. Representative FACS dot blots from spleen (a–g) and mesenteric lymph nodes (mLN) (h–n) are shown. No significant differences were observed either in spleen or mLN between infective larvae of B. malayi (Bm-L3), recombinant Wolbachia surface protein (r-wsp) and Bm-L3+r-wsp treated mice in comparison with control. The dot plots are representative of three independent experiments performed on different days with five or six animals per group.

polarized Th1 response in BALB/c mice (resistant host) after administering r-wsp, which may facilitate B. malayi L3 establishment following challenge. It has been reported that r-wsp immunization with Freund’s complete adjuvant leads to enhanced establishment of L3 of Litomosoides sigmodontis in BALB/c mice compared with unvaccinated controls.45 In this context, we may presume that in a filaria-endemic area exposure of human hosts to WSP as a result of natural worm death or anti-filarial chemotherapy may facilitate the establishment of infective larvae inoculated by infected mosquitoes. While Treg cells are known regulators of Th17 responses in mice and reciprocal regulation of Th17 and Treg cells is an established feature of T-cell differentiation, which is governed by the presence of specific cytokines,46,47 e.g. TGF-b can induce both Treg cells and Th17 cells. The presence of accessory cytokines IL-6 or IL-21, however, skews the development of Th17 cells at the expense of the Treg cell lineage.48,49 This interdependent regulation of Th17 and Treg cells is less well-characterized in humans than is the association of Th17 up-regulation and Treg suppression that has been described in only a few human autoimmune and inflammatory disorders. Our study therefore reveals that, in an infectious disease setting of filariasis, there is a clear inverse relationship between Th17 cells and Treg cells and the balance between Th17 responses and Treg cells might play a vital role in mediating the progress of infection. However, recently it has been shown that FoxP3+ Treg cells can acquire the ability to produce IL-17, suggesting that Treg cells can potentially contribute to the antimicrobial innate immune defence while controlling inflammation and autoimmunity at the same time, particularly at mucosal sites. Interferon-c, a major product of Th1 cells, is critical for the initiation of cell-mediated immunity against intracellular pathogens and in the induction of some autoimmune diseases.50,51 Interestingly, many IFN-producing Th1 cells have been found to produce IL-10, an anti-inflammatory cytokine, in antimicrobial immune responses.52 This phenomenon, known as selfcontrol of Th1 cells, is a critical mechanism for effective antimicrobial immune responses while limiting self-tissue damage.52 The identification of IL-17-producing Treg cells in both mice and humans suggests that Th17 and Treg cell lineages are related in ontogeny and both lineages appear to depend on TGF-b for their differentiation and/or main241

M. Pathak et al. Spleen 2·48%

104

103

103

2

10 0

0

3

4

10 10 10

5

2

0 10 (i) 105

Q2

Q1 4·77%

0·98%

104

104

3

10

103

102 0

102 0

Q3 20·5%

FMO

Q5 0·331%

(j)

Q6

0·36%

104

103

103

2

2

Adjuvant

(d) 105

Q2

Q1 1·81%

105 104

103

103

L3

103

103

0

Q2

Q1 1·38%

1·65%

(m) 5 10 104

103

103

2

0 10 (g) 105

3

4

Q2

1·63%

105 104

103

103 Q3 31·9%

0

102

103

4

10

Q2

3

Q3 18·8% 4 5

10 10 10

Q1 3·83%

Q2

2·50%

Q3 21·6%

0

102

103

Q1 2·64%

104

105 Q2

0·99%

3

Q3 21·5% 4 5

2000 1000

Q2

Q3 15·9%

0

102

Figure 10. Estimation of transforming growth factor-b (TGF-b) in secondary lymphoid organ of mice. TGF-b was estimated in culture supernatant of splenocytes from mice of different treated groups by ELISA as described in Materials and methods. TGF-b concentration (pg/ml) obtained in different groups is shown. Data represents mean  SEM values from three separate experiments (n = 5–6 in each group). Statistical significance between treated and control groups is depicted as ***P ≤ 0001.

Taken together, our results show that r-wsp acts synergistically with Bm-L3 in promoting pro-inflammatory response by Th17 cells and at the same time diminishes host immunological tolerance by decreasing Treg cells and TFG-b. The results highlight the synergistic action of r-wsp and Bm-L3 in reducing Treg cells and TFG-b from secondary lymphoid organs of infected mice, leading to diminished immunological tolerance and at the same time promoting a pro-inflammatory response that is characterized by attenuated Th2 response as well as increased percentages of Th1 cytokines and Th17 cells in the spleens and mLN of infected animals.

Acknowledgements

1·00%

103

104

We are grateful to Mr A.K. Roy for his excellent technical support in maintaining B. malayi infection in the laboratory and Mr A.L. Vishwakarma for assisting with flow cytometry experiments.

105

CD4

tenance, and additional cytokines may determine whether they become Th17, Treg or dual-function effector T cells.53 Foxp3+ Treg cells may therefore actively contribute to antimicrobial innate immunity by producing IL-17, whereas they control inflammation and autoimmunity at the same time. 242

3000

cont adj L3 r-wsp r-wsp + L3

10 10 10

Q1 2·51%

102 0

105

5

1·57%

0 10 (n)

104

102 0

4

10 10 10

Q1 3·14%

2

5

10 10 10

Q1 1·49%

Q7 20·9% 3

102 0

Q3 33·5%

*** *** ***

Animals groups

102 0

104

102 0

r-wsp + L3

105 104

(f) 105

r-wsp

2·38%

Q3 43·3% 2 3 4 10 10 10 105

Q6

0·25%

4000

0

0 10 (l)

104

102 0

Q5 0·105%

2

0 102 103 104 105 Q2

Q3 21·0%

102 0

Q3 41·4%

Q1 1·41%

5000

0 10 (k)

104

(e) 105

Q2

Figure 9. Estimation of CTLA-4 in secondary lymphoid organs of mice CTLA-4 was estimated by flow cytometry as described in Materials and methods. Representative FACS dot blots from spleen (a–g) and mesenteric lymph nodes (mLN) (h–n) are shown. No significant differences were observed either in spleen or mLN between infective larvae of B. malayi (Bm-L3), recombinant Wolbachia surface protein (r-wsp) and Bm-L3+r-wsp treated mice in comparison with control. The dot plots are representative of three independent experiments performed on different days with five or six animals per group.

0·37%

2

2·28%

102 0

Q3 23·0% 4 105

10 10

Q1 1·44%

10 0

Q7 20·5%

0 102 103 104 105

CTLA4

105

104

10 0

3

0 102 103 104 105

0 102 103 104 105 (c) 105

2·37%

102 0

Q3 33·4%

102

Q2

105 Q1 2·40%

104

(b) 105

Isotype

mLN (h)

Q2

Q1 2·39%

TGF-β (pg/ml)

Control

(a) 105

Author contribution MP, MV and MS performed the experiments, MP, MS and SMB designed the study, MP, MS and SMB wrote the paper.

Funding The financial support was received in the form of research fellowships to the research students from the ª 2014 John Wiley & Sons Ltd, Immunology, 144, 231–244

WSP mediates pro-inflammatory response Indian Council of Medical Research (ICMR), New Delhi, India. The authors also wish to acknowledge funding support provided to MS and SMB via the CSIR-Network project SPlenDID and UNDO. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This manuscript bears the CDRI communication no. 8739.

Disclosures The authors declare no conflicts of interest.

References 1 Werren JH, Baldo L, Clark ME. Wolbachia: master manipulators of invertebrate biology. Nat Rev Microbiol 2008; 6:741–51. 2 Brattig NW, Buttner DW, Hoerauf A. Neutrophil accumulation around Onchocerca worms and chemotaxis of neutrophils are dependent on Wolbachia endobacteria. Microbes Infect 2001; 3:439–46. 3 Hoerauf A, Volkmann L, Nissen-Paehle K et al. Targeting of Wolbachia endobacteria in Litomosoides sigmodontis: comparison of tetracyclines with chloramphenicol, macrolides and ciprofloxacin. Trop Med Int Health 2000; 5:275–9. 4 Casiraghi M, McCall JW, Simoncini L et al. Tetracycline treatment and sex-ratio distortion: a role for Wolbachia in the moulting of filarial nematodes? Int J Parasitol 2002; 32:1457–68. 5 Taylor MJ, Bandi C, Hoerauf A. Wolbachia bacterial endosymbionts of filarial nematodes. Adv Parasitol 2005; 60:245–84. 6 Bazzocchi C, Mortarino M, Grandi G et al. Combined ivermectin and doxycycline treatment has microfilaricidal and adulticidal activity against Dirofilaria immitis in experimentally infected dogs. Int J Parasitol 2008; 38:1401–10. 7 Hoerauf A. Filariasis: new drugs and new opportunities for lymphatic filariasis and onchocerciasis. Curr Opin Infect Dis 2008; 21:673–81. 8 Specht S, Wanji S. New insights into the biology of filarial infections. J Helminthol 2009; 83:199–202. 9 Supali T, Djuardi Y, Pfarr KM et al. Doxycycline treatment of Brugia malayi-infected persons reduces microfilaremia and adverse reactions after diethylcarbamazine and albendazole treatment. Clin Infect Dis 2008; 46:1385–93. 10 Mand S, Pfarr K, Sahoo PK et al. Macrofilaricidal activity and amelioration of lymphatic pathology in Bancroftian filariasis after 3 weeks of doxycycline followed by singledose diethylcarbamazine. Am J Trop Med Hyg 2009; 81:702–11. 11 Turner JD, Langley RS, Johnston KL, Egerton G, Wanji S, Taylor MJ. Wolbachia endosymbiotic bacteria of Brugia malayi mediate macrophage tolerance to TLR- and CD40-specific stimuli in a MyD88/TLR2-dependent manner. J Immunol 2006; 177:1240–9. 12 Sakaguchi S. Naturally arising CD4+ regulatory t cells for immunologic self-tolerance and negative control of immune responses. Annu Rev Immunol 2004; 22:531–62. 13 Taylor MJ, Cross HF, Bilo K. Inflammatory responses induced by the filarial nematode Brugia malayi are mediated by lipopolysaccharide-like activity from endosymbiotic Wolbachia bacteria. J Exp Med 2000; 191:1429–36. 14 Gillette-Ferguson I, Hise AG, McGarry HF et al. Wolbachia-induced neutrophil activation in a mouse model of ocular onchocerciasis (river blindness). Infect Immun 2004; 72:5687–92. 15 Babu S, Nutman TB. Proinflammatory cytokines dominate the early immune response to filarial parasites. J Immunol 2003; 171:6723–32. 16 Melnikow E, Xu S, Liu J, Li L, Oksov Y, Ghedin E, Unnasch TR, Lustigman S. Interaction of a Wolbachia WSP-like protein with a nuclear-encoded protein of Brugia malayi. Int J Parasitol 2011; 41:1053–61. 17 Brattig NW, Bazzocchi C, Kirschning CJ et al. The major surface protein of Wolbachia endosymbionts in filarial nematodes elicits immune responses through TLR2 and TLR4. J Immunol 2004; 173:437–45. 18 Lin X, Xu L, Feng Y, Li J, Pei L, Shen T, Shao Y. Clinical study on treatment of HIV infected persons based on viral load detection and CD4+ T lymphocyte counting. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi 2002; 16:211–4. 19 Ghedin E, Daehnel K, Foster J, Slatko B, Lustigman S. The symbiotic relationship between filarial parasitic nematodes and their Wolbachia endosymbionts: a resource for a new generation of control measures. Symbiosis 2008; 46:77–85. 20 Bazzocchi C, Comazzi S, Santoni R, Bandi C, Genchi C, Mortarino M. Wolbachia surface protein (WSP) inhibits apoptosis in human neutrophils. Parasite Immunol 2007; 29:73–9.

ª 2014 John Wiley & Sons Ltd, Immunology, 144, 231–244

21 Hise AG, Daehnel K, Gillette-Ferguson I et al. Innate immune responses to endosymbiotic Wolbachia bacteria in Brugia malayi and Onchocerca volvulus are dependent on TLR2, TLR6, MyD88, and Mal, but not TLR4, TRIF, or TRAM. J Immunol 2007; 178:1068–76. 22 Lawrence RA, Allen JE, Gregory WF, Kopf M, Maizels RM. Infection of IL-4-deficient mice with the parasitic nematode Brugia malayi demonstrates that host resistance is not dependent on a T helper 2-dominated immune response. J Immunol 1995; 154:5995– 6001. 23 Balmer P, Devaney E. NK T cells are a source of early interleukin-4 following infection with third-stage larvae of the filarial nematode Brugia pahangi. Infect Immun 2002; 70:2215–9. 24 Ramalingam T, Rajan B, Lee J, Rajan TV. Kinetics of cellular responses to intraperitoneal Brugia pahangi infections in normal and immunodeficient mice. Infect Immun 2003; 71:4361–7. 25 Shiny C, Krushna NS, Archana B, Farzana B, Narayanan RB. Serum antibody responses to Wolbachia surface protein in patients with human lymphatic filariasis. Microbiol Immunol 2009; 53:685–93. 26 Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72:248–54. 27 Kushwaha S, Singh PK, Rana AK, Misra-Bhattacharya S. Cloning, expression, purification and kinetics of trehalose-6-phosphate phosphatase of filarial parasite Brugia malayi. Acta Trop 2011; 119:151–9. 28 Kushwaha S, Singh PK, Gupta J, Soni VK, Misra-Bhattacharya S. Recombinant trehalose-6-phosphate phosphatase of Brugia malayi cross-reacts with human Wuchereria bancrofti immune sera and engenders a robust protective outcome in mice. Microbes Infect 2012; 14:1330–9. 29 Singh M, Singh PK, Misra-Bhattacharya S. RNAi mediated silencing of ATPase RNA helicase gene in adult filarial parasite Brugia malayi impairs in vitro microfilaria release and adult parasite viability. J Biotechnol 2012; 157:351–8. 30 Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(–DDCT) method. Methods 2001; 25:402–8. 31 Babu S, Bhat SQ, Pavan KN, Lipira AB, Kumar S, Karthik C, Kumaraswami V, Nutman TB. Filarial lymphedema is characterized by antigen-specific Th1 and Th17 proinflammatory responses and a lack of regulatory T cells. PLoS Negl Trop Dis 2009; 3:21. 32 Suba N, Shiny C, Taylor MJ, Narayanan RB. Brugia malayi Wolbachia hsp60 IgG antibody and isotype reactivity in different clinical groups infected or exposed to human Bancroftian lymphatic filariasis. Exp Parasitol 2007; 116:291–5. 33 Harrington LE, Hatton RD, Mangan PR, Turner H, Murphy TL, Murphy KM, Weaver CT. Interleukin 17-producing CD4+ effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages. Nat Immunol 2005; 6:1123–32. 34 Korn T, Bettelli E, Oukka M, Kuchroo VK. IL-17 and Th17 cells. Annu Rev Immunol 2009; 27:485–517. 35 Braig HR, Zhou W, Dobson SL, O’Neill SL. Cloning and characterization of a gene encoding the major surface protein of the bacterial endosymbiont Wolbachia pipientis. J Bacteriol 1998; 180:2373–8. 36 Baldo L, Lo N, Werren JH. Mosaic nature of the Wolbachia surface protein. J Bacteriol 2005; 187:5406–18. 37 Baldo L, Desjardins CA, Russell JA, Stahlhut JK, Werren JH. Accelerated microevolution in an outer membrane protein (OMP) of the intracellular bacteria Wolbachia. BMC Evol Biol 2010; 10:1471–2148. 38 Serbus LR, Casper-Lindley C, Landmann F, Sullivan W. The genetics and cell biology of Wolbachia–host interactions. Annu Rev Genet 2008; 42:683–707. 39 Taylor MD, van der Werf N, Harris A, Graham AL, Bain O, Allen JE, Maizels RM. Early recruitment of natural CD4+ Foxp3+ Treg cells by infective larvae determines the outcome of filarial infection. Eur J Immunol 2009; 39:192–206. 40 Liew FY, Xu D, Brint EK, O’Neill LA. Negative regulation of toll-like receptor-mediated immune responses. Nat Rev Immunol 2005; 5:446–58. 41 Yazdanbakhsh M, Sartono E, Kruize YC et al. Elevated levels of T cell activation antigen CD27 and increased interleukin-4 production in human lymphatic filariasis. Eur J Immunol 1993; 23:3312–7. 42 Bennuru S, Semnani R, Meng Z, Ribeiro JM, Veenstra TD, Nutman TB. Brugia malayi excreted/secreted proteins at the host/parasite interface: stage- and gender-specific proteomic profiling. PLoS Negl Trop Dis 2009; 3:e410. 43 McSorley HJ, Harcus YM, Murray J, Taylor MD, Maizels RM. Expansion of Foxp3+ regulatory T cells in mice infected with the filarial parasite Brugia malayi. J Immunol 2008; 181:6456–66. 44 Ravindran B. Are inflammation and immunological hyperactivity needed for filarial parasite development? Trends in Parasitol 2001; 17:70–3. 45 Lamb TJ, Harris A, Le Goff L, Read AF, Allen JE. Litomosoides sigmodontis: vaccineinduced immune responses against Wolbachia surface protein can enhance the survival of filarial nematodes during primary infection. Exp Parasitol 2008; 118:285–9.

243

M. Pathak et al. 46 Leoratti FM, Durlacher RR, Lacerda MV, Alecrim MG, Ferreira AW, Sanchez MC, Moraes SL. Pattern of humoral immune response to Plasmodium falciparum blood stages in individuals presenting different clinical expressions of malaria. Malar J 2008; 7:1475–2875. 47 Robinson LJ, D’Ombrain MC, Stanisic DI et al. Cellular tumor necrosis factor, gamma interferon, and interleukin-6 responses as correlates of immunity and risk of clinical Plasmodium falciparum malaria in children from Papua New Guinea. Infect Immun 2009; 77:3033–43. 48 Mahanty S, Ravichandran M, Raman U, Jayaraman K, Kumaraswami V, Nutman TB. Regulation of parasite antigen-driven immune responses by interleukin-10 (IL-10) and IL-12 in lymphatic filariasis. Infect Immun 1997; 65:1742–7. 49 Fouser LA, Wright JF, Dunussi-Joannopoulos K, Collins M. Th17 cytokines and their emerging roles in inflammation and autoimmunity. Immunol Rev 2008; 226:87–102. 50 Haskins K, McDuffie M. Acceleration of diabetes in young NOD mice with a CD4+ islet-specific T cell clone. Science 1990; 249:1433–6. 51 Sadick MD, Heinzel FP, Shigekane VM, Fisher WL, Locksley RM. Cellular and humoral immunity to Leishmania major in genetically susceptible mice after in vivo depletion of L3T4+ T cells. J Immunol 1987; 139:1303–9. 52 Trinchieri G. Interleukin-10 production by effector T cells: Th1 cells show self control. J Exp Med 2007; 204:239–43. 53 Zhou L, Lopes JE, Chong MM et al. TGF-b-induced Foxp3 inhibits TH17 cell differentiation by antagonizing RORct function. Nature 2008; 453:236–40.

Supporting Information Additional Supporting Information may be found in the online version of this article:

244

Figure S1. Real time RT-PCR analysis of T helper type 1 (Th1) and Th2 cytokines. Figure S2. Intracellular interleukin-17 (IL-17) release was measured in the purified CD4+ T-cell fraction using flow cytometry as described in Materials and methods. Figure S3. Mice were immunized with recombinant trehalose-6-phosphate phosphatase protein (r-tpp) and subsequently infected with infective larvae of B. malayi (Bm-L3), flow cytometric analysis of T helper type 17 (Th17) cells was performed as described in Materials and Methods. Figure S4. Regulatory T (Treg) cells were measured in the purified CD4+ T-cell fraction using flow cytometry as described in the Materials and methods. Figure S5. Intracellular cytokine production was measured by flow cytometry for various cytokines as described in the Materials and methods. Table S1. Primer sequences used for Real time RT-PCR analysis.

ª 2014 John Wiley & Sons Ltd, Immunology, 144, 231–244