- Email: [email protected]
Ras isoforms selectively regulate antigen-specific immune response
Cytokine 126 (2020) 154914
Contents lists available at ScienceDirect
Cytokine journal homepage: www.elsevier.com/locate/cytokine
Short communication
Ras isoforms selectively regulate antigen-specific immune response ⁎
T
Mukesh Kumar Jha, Aditya Y. Sarode, Bhaskar Saha National Centre for Cell Science, Ganeshkhind, Pune 411007, India
A R T I C LE I N FO
A B S T R A C T
Keywords: Ras isoforms Leishmania TNP-Ova Ras GTPase Cytokine
H-/K-Ras and N-Ras isoforms were proposed to lack functional specificities due to similarity in 1–165 amino acids. As recent studies implied Ras isoform-specific developmental effects, we examined their functional specificity using Leishmania major infection, anti-hapten antibody response and carrier-specific T cell response. While N-Ras overexpression increased L. major infection in resistant C57BL/6 mice, H-Ras or K-Ras overexpression reduced the infection in susceptible BALB/c mice. These Ras isoforms differentially regulated antiTNP antibody response in TNP-Ova-primed, but not in TNP-Ficoll- or TNP-LPS-primed, BALB/c mice. Ras isoform-specific silencing selectively modulated Ova-specific T cell response. The data indicate Ras isoform-specific regulation of antigen-specific immune response.
1. Introduction Antigen-specific immune responses are regulated by many factors including cell signaling intermediates. One of these signaling intermediates is Ras GTPases. Ras GTPases have three major isoforms- NRas, K-Ras and H-Ras. Ras-GTPases were chiefly implicated in oncogenesis and development [1]. Owing to the high sequence homology in their N-terminal 1–165 amino acid residues [2], these ubiquitously expressed Ras isoforms are considered to be functionally redundant [3]. However, an association of K-Ras and N-Ras mutations with pancreatic cancers and leukemia, respectively [4], and indispensable role of K-Ras, but not H-Ras or N-Ras, in mouse development [5] implied non-redundancy among Ras isoforms. These studies focused on the role of Ras in oncogenesis or development but the role of Ras isoforms in modulating antigen-specific immune response is not yet explicitly demonstrated. Therefore, we examined the Ras isoforms’ role in the modulation of antigen-specific immune responses to 2, 4, 6-TrinitrophenylOvalbumin (TNP-Ova), as compared with T-independent antigensTNP-Ficoll and TNP-LPS- as an internal control for antigen-processing and T-dependent immune response. H/-K-Ras overexpression in a susceptible host or N-Ras overexpression in a resistant mouse turns them resistant or susceptible, respectively, to L. major infection. The selective modulation of the anti-TNP antibody response to TNP-Ova by the Ras isoforms matches the profile with the infection indicating Ras isoformspecific regulation of antigen-specific immune response. Anti-TNP
IgG2a production corroborated with IFN-γ production from the ovalbumin-stimulated T-cells in TNP-Ova-primed mice. These data indicate Ras isoform-specific functions paving the way for Ras-targeted therapy against many diseases. 2. Materials and methods 2.1. Reagents Immunization antigens TNP-Ova, TNP-Ficoll, TNP-LPS and TNPBSA (Biosearch Technology, Inc, CA) and EndoFit Ovalbumin (InvivoGen, San Diego, CA). Western blot antibodies for N-Ras, K-Ras, H-Ras, β-actin and Luminol reagent were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-cytokine Abs (IFN-γ, and IL-4) and cytokine standard for ELISA were purchased from BD Biosciences (San Diego, CA). FACS antibodies anti-CD3-PE-Cy7, anti-CD25-APCCy7 and anti-IL-10-FITC (BD Biosciences Pharmingen, San Diego, CA), anti-CD4-PerCP-Cy5.5 and anti-Foxp3-Pacific Blue (BioLegend, San Diego, CA) and anti-CD127-APC (eBioscience, Santa Clara, CA) were purchased. 2.2. Animals, cell line, parasites and infection BALB/c and C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME) were bred in the experimental animal facility at the National Centre for
Abbreviations: TNP-Ova, 2, 4, 6-Trinitrophenyl-Ovalbumin; TNP-Ficoll, 2, 4, 6-Trinitrophenyl-AECM-Ficoll; TNP-LPS, 2, 4, 6-Trinitrophenyl -Lipopolysaccharide; TNP-BSA, 2, 4, 6-Trinitrophenyl-Bovine Serum Albumin; IFA, Incomplete Freund’s adjuvant; IP, Intra peritoneal; SC, Subcutaneous; Ctrl, Control; CV, Control Vector; Inf, Infection ⁎ Corresponding author. E-mail address: [email protected] (B. Saha). https://doi.org/10.1016/j.cyto.2019.154914 Received 2 August 2019; Received in revised form 5 October 2019; Accepted 30 October 2019 1043-4666/ © 2019 Published by Elsevier Ltd.
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maintained by passage through BALB/c mice. Stationary-phase L. major promastigotes (2x106/mouse, s.c. in the hind footpad) were used to infect BALB/c and C57BL/6 mice. Mice were treated s.c in the hind footpad with 5 × 106 transduction units of lentivirus-expressing N-Ras, K-Ras, H-Ras, pGIPZ, pLKO.1 or control empty vector 2 days prior to infection.
Cell Science in Thoren caging units (Thoren Caging System, Hazleton, PA). The progress of infection was monitored weekly by measuring footpad thickness and parasite load was assessed following euthanization five weeks after the infection. All experiments were in accordance with the animal use protocol approved by the Institutional Animal Care and Use Committee and the Committee for the Purpose of Control and Supervision of Experiments on Animals, the regulatory authorities for animal experimentation. P388D1, a macrophage-like cell line, was obtained from ATCC (American Type Culture Collection) and cultured in RPMI-1640 medium (Life Technologies-BRL) containing penicillin (70 µg/ml), streptomycin (100 µg/ml), 2-mercaptoethanol (50 µM), sodium pyruvate (1 µM), HEPES (20 µM) and 10% heat inactivated FCS (Life Technologies-BRL). L. major (strainMHOM/Su73/5ASKH) was maintained in vitro in RPMI 1640 medium containing penicillin (70 µg/ ml), streptomycin (100 µg/ml), 2-mercaptoethanol (50 µM), sodium pyruvate (1 µM), supplemented with 10% FCS and the virulence was
2.3. Peritoneal macrophage collection C57BL/6 female mice were injected with 2 ml thioglycolate (3% i.p.). Four days later, peritoneal exudate cells were harvested in sterile HBSS (Life Technologies-BRL), centrifuged (210g, 8 min at 4 °C), and the pellet was resuspended in RPMI 1640 with 10% FCS. The cells were seeded in 30 mm culture plates and were cultured at 37 °C in a humidified CO2 incubator for 12 h. Non-adherent cells were removed by replacing the medium with fresh RPMI 1640 with 10% FCS. The culture
Fig. 1. Ras isoforms show specificity in modulating Leishmania major infection in BALB/c and C57BL/6 mice and antigen-specific Ras isoforms modulate T-reg numbers. (a. Left panel) Five BALB/c mice/group were treated with overexpressed Ras isoforms or control vector 2 days prior to infection. Two days later BALB/c mice were infected s.c. in hind footpad with 2 × 106 L. major stationary promastigotes. Disease progression was scored weekly by evaluating the net footpad swelling (the thickness of the uninfected left footpad subtracted from the infected right footpad) by digital micrometer (Mitutoyo Japan) for 5 weeks. The Ras isoforms gene silencing was confirmed (insert, 1a). (a. Middle panel) BALB/c mice were sacrificed 5 weeks after the infection, and parasite burden in lymph node cells was assessed. (a. Right panel) Lymph node weight in treated and infected parasite. (b. Left panel) C57BL/6-derived peritoneal macrophages were treated with N-Ras overexpressed or control vector (2 MoI; 48 h), followed by pulsing with purified M10 (10 µg/ml; 6 h). The macrophages were washed and injected s.c. into the hind footpad of C57BL/6 mice (106 macrophages/mouse) weekly for 3 wks. Four weeks later, the mice were challenged with 2 × 106 L. major stationary promastigotes (s.c.) in the hind footpad. Disease progression was scored weekly by evaluating the net footpad swelling (the thickness of the uninfected left footpad subtracted from the infected right footpad) by digital micrometer for 5 weeks. The Ras isoforms gene overexpression was confirmed (insert, 1b). (b. Middle panel) C57BL/6 mice were sacrificed 5 wks after the infection, and parasite burden in lymph node cells was assessed. (b. Right panel) Lymph node weight in treated and infected parasite. The experiments were performed twice and one representative data are shown. Error bars represent mean ± SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. Five BALB/c mice/group were treated with (c) silenced Ras isoform and (d) overexpressed Ras isoforms along with control vector 2 days prior to immunization (s.c with 60 µg TNP-Ova/mouse in IFA) and 21 days after the immunization, splenic T cells were collected. Five mice per group were sacrificed and pooled for FACS staining without any stimulation. T cells were stained with T-reg cell-specific antibodies were acquired on CD3+CD4+CD25+CD127dim gating and analyzed for expression of IL-10 vs Foxp3, as described in Materials and Methods. The values inside graphs represent % of cells. The experiments were performed twice, and one representative data are shown. 2
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2.8. Production of lentiviral particles for Ras isoforms shRNA and control shRNA
was maintained as per the requirements of the experiments. 2.4. Western blotting
A third generation SIN (self-inactivating) lentiviral vector-based system was used. N-Ras construct in pGIPZ vector, H-Ras and K-Ras constructs in pLKO.1 vector and the corresponding scrambled sequence for control constructs were from Open Biosystems (Huntsville, AL). Using a lentiviral packaging system, we generated replication incompetent HIV-1 based lentivirus for all isoform-specific and control constructs. The viral titer was assessed and the virus particles were concentrated according to the manufacturer’s protocol [6].
Cells were washed twice with chilled PBS and lysed in cell lysis buffer (20 mM Tris [pH 7.4], 150 mM NaCl, 1% Nonidet P-40, 10% glycerol, 2 mM EDTA, protease inhibitor mixture [Roche Applied Science, Mannheim, Germany], and phosphatase inhibitor mixture [Pierce, Rockford, IL]). Protein was quantified by a BCA kit (Pierce) or Bradford reagent and an equal amount of protein was run on SDSPAGE. Resolved proteins were blotted onto polyvinylidene difluoride (Millipore, Bedford, MA) and blocked with 5% nonfat dried milk in TBST (25 mM Tris [pH 7.6], 137 mM NaCl, and 0.2% Tween-20). Membranes were incubated with primary Ab at 4 °C overnight, washed with TBST, and incubated with HRP-conjugated secondary Ab. Immunoreactive bands were visualized with the chemiluminescent Luminol reagent (Santa Cruz Biotechnology, Santa Cruz, CA) (6).
2.9. Statistical analyses The significance of difference between the means was calculated from Student’s t-test. The minimum number of mice (female 6–8 weeks old) used for in vivo experiments was five per group. The results were plotted as mean ± standard error of means (SEM) from triplicate sets of experiments. The significance of difference between the means was considered statistically significant when *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.
2.5. FACS-Treg cell analyses T cells from naive and TNP Ova treated mice were studied for Treg cells by multicolour FACS analyses. After blocking with 5% FCS for 30 min at 4 °C, the cells were washed twice with FACS buffer (1XPBS, 10 mM HEPES buffer and 3% FCS) and stained with anti-CD3-PE-Cy7, anti-CD4- PerCP-Cy5.5, anti-CD127-APC and anti-CD25-APC-Cy7 (45 min, 4 °C) and washed twice with FACS buffer. For intracellular staining, the cells were permeabilized using Cytofix/Cytoperm-Plus Kit with GolgiPlug and washed with Perm/Wash buffer. The cells were stained with anti-Foxp3-Pacific Blue, anti-IL-10-FITC antibodies (60 min, 4 °C). Cells were acquired in the CD3+CD4+CD25+CD127dim gate for Treg cells in a FACS CantoII flow cytometer and analyzed for expression of Foxp3 and IL-10 using BD-FACS Diva software (v5.2; BD Biosciences). Cells stained with Isotype-specific antibodies were used as controls.
3. Results 3.1. Ras isoforms overexpression can dictate susceptibility vs resistance in Leishmania infection While Ras isoforms expression and activities in macrophages were differentially regulated by L. major infection [6], higher L. major infection was reported in N-Ras- and H-Ras-deficient L. major-resistant C57BL/6 mice [7]. We overexpressed H-, K-, or N-Ras isoforms and control vector in susceptible BALB/c mice, followed by L. major infection and assessment of the foot-pad thickness (Fig. 1a Left panel), parasite load (Fig. 1a Middle panel), and lymph node weight (Fig. 1a Right panel). We observed that H-/K-Ras overexpression reduced, whereas N-Ras overexpression increased the infection in BALB/c mice (Fig. 1a, left and middle panels) accompanied by draining lymph node hypertrophy (Fig. 1a, right panel). We further overexpressed N-Ras in C57BL/6-derived elicited macrophages that are pulsed with purified L. major MAPK10 (M10) [8]. These macrophages were reintroduced into C57BL/6 mice followed by challenge with L. major. We observed that the recipient L. major-resistant mice were turned susceptible to infection showing significantly higher parasite burden (Fig. 1b). These data indicate that N-Ras overexpression favours L. major infection whereas K-Ras and H-Ras overexpression inhibits L. major infection.
2.6. Sandwich ELISA for quantifying cytokines The culture supernatants from the splenic T cells stimulated with the indicated doses of Ova for 48 h were assayed for IFN-γ or IL-4 by ELISA [6]. Briefly, ELISA plates were coated overnight at 4 °C with purified Abs to IL-4 (1 µg/ml), or IFN-γ (2 µg/ml). Plates were washed thrice (0.05% Tween 20 in 1X PBS) and blocked for 2 h with 1% BSA. Plates were incubated overnight with cytokine standards or culture supernatants. Plates were washed and incubated with respective biotinconjugated detection Abs for 1 h at 25 °C. Plates were washed and incubated with peroxidase-conjugated streptavidin (Roche Applied Science) for 30 min followed by washing and development with tetramethylbenzidine substrate (BD Pharmingen, San Diego, CA). Reaction was stopped by the addition of 1 N H2SO4, and absorbance was measured at 450 nm in an ELISA reader (VERSA max microplate reader, Molecular Devices). The values of standards were plotted and the quantities of the cytokines were estimated in the samples and expressed in pg/mL.
3.2. Ras isoforms-specific regulation of T-reg cells T-regulatory (T-reg) cells were proposed as professional suppressors of immune responses and, in corroboration with that proposition; these cells were implicated in the persistence of Leishmania parasite in a susceptible host [9–11]. As T-reg cells expansion may be dependent on the state of APC activation that, in turn, can be controlled by Ras isoforms, we examined whether silencing of Ras isoforms individually could affect the T-reg cells in Ova-primed BALB/c mice. Therefore, we administered lentivirally-expressed N-Ras shRNA, K-Ras shRNA or HRas shRNA or control shRNA and overexpressed Ras isoforms or control vector into Ova-primed BALB/c mice. The mice were sacrificed 21 days after the priming and the T-reg cells were enumerated by multi-color FACS. We observed higher IL-10 on Foxp3-gated cells in K-Ras and HRas, but not N-Ras (Fig. 1c), silenced condition, whereas Ras isoforms overexpression reversed the phenomena in Ova-primed BALB/c mice (Fig. 1d). These data suggest that Ras isoforms contribute to T-reg cells response.
2.7. Antibody ELISA To detect antigen-specific immunoglobulin, 96 well microtiter plates were coated with 2,4,6-Trinitrophenyl-Bovine Serum Albumin (TNP-BSA-10 µg/ml) at 4 °C (overnight). Using 1% BSA (in 1X PBS), the plates were blocked for 2 h at room temperature and sera (dilutions prepared with the help of 1X PBS) from different groups of mice were added to the coated plates and incubated overnight at 4 °C. The plates were washed four times with wash buffer and bound antibodies were detected using biotin-conjugated goat anti-mouse IgG1, IgG2a and IgM specific antibodies (BD Bioscience; San Diego, CA). Antibody isotypes were measured by ELISA [6]. 3
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Fig. 2. Functional specificities of Ras isoforms in the TNP-Ova primed BALB/c mice. (a). Five female BALB/c mice/group were treated with Ras isoform shRNA Lv or control shRNA Lv 2 days prior to immunization (s.c with 60 µg TNP-Ova, 10 µg TNP-LPS and 50 µg TNP-Ficoll /mouse in IFA) and 10 days after the immunization, sera were collected and checked for the levels of IgG1, IgG2a and IgM antigen-specific immunoglobulin by indirect antibody ELISA method, as described in Materials and Methods. (b) BALB/c mice were treated with overexpressed Ras isoforms or control vector 2 days prior to immunization (s.c with 60 µg TNP-Ova, 10 µg TNP-LPS and 50 µg TNP-Ficoll /mouse in IFA). 10 days after the immunization and checked for the levels of IgG1, IgG2a and IgM antigen-specific immunoglobulin by indirect antibody ELISA in the sera as described in Materials and Methods. Post 21 days of immunization, (c) Ras isoforms silenced or (d) Ras isoforms overexpressed and TNP-Ova immunized mice were sacrificed and collected the splenic T-cells and cultured in presence of the indicated doses of Ova for 48 h. Culture supernatants were assessed for the production of the cytokines IFN-γ and IL-4, as described in Materials and Methods. The experiments were performed thrice, and one representative data are shown. Error bars represent mean ± SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.
3.3. Ras isoforms selectively modulate antigen-specific immune response
4. Discussion
Lentiviral expression of H-Ras or K-Ras shRNA reduced TNP-specific IgG2a antibody whereas N-Ras silencing reduced anti-TNP IgG1 isotype in TNP-Ova primed mice (Fig. 2a and Supplementary Fig. 1a). Ras isoforms overexpression reversed the phenomena (Fig. 2b and Supplementary Fig. 1b). Ova-specific T cell response corroborated with the isotype profile, as H-Ras or K-Ras silencing reduced IFN-γ production whereas N-Ras silencing reduced IL-4 production (Fig. 2c). A reverse profile was obtained in Ras isoforms overexpression (Fig. 2d) indicating the role of Ras isoforms functional specificities in an antigen-specific immune response. Contrary to the effects of TNP-OVA, Ras isoforms silencing or overexpression had little effects on anti-TNP isotypes in response to TNP-LPS (Fig. 2a-b and Supplementary Fig. 2a-b) or TNPFicoll (Fig. 2a-b and Supplementary Fig. 3a-b) immunization. LPS and Ficoll being T-independent antigens, the TNP-specific IgM did not switch to IgG1 or IgG2a. Anti-TNP IgM antibody however switched to IgG1 or IgG2a depending on Ras isoforms manipulated suggesting that Ras isoforms effect on antibody is probably through T cells or antigenpresenting cells. Although Ras isoforms were previously implicated in differential regulation of proliferation, anergy and positive and negative selection of T-cells in thymus [12–15], cellular signaling [16] and in tissue-specific carcinogenesis, the functional specificities are assigned to Ras isoforms by the antigen-specific immune response reported here. Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.cyto.2019.154914.
Our data show how specific Ras isoforms can be targeted for antileishmanial immunotherapy and immunoprophylaxis. The roles played by Ras isoforms in antigen-specific immune response were not previously documented. Involvement of Ras isoforms in CD40 signaling in peritoneal macrophages was indicated [6] but herein, the role of individual Ras isoforms in T cell or B cell response using T- dependent and T-independent hapten-carrier conjugates- TNP-Ova, TNP-Ficoll and TNP-LPS and the role of each Ras isoform in anti-leishmanial immune response have been studied. Both Leishmania infection and the T-dependent hapten-carrier (TNP-Ova) response models show Ras isoformspecific modulation of T cell response. As silencing of N-Ras resulted in reduced carrier-specific T cell’s IL-4 production but K-Ras or H-Ras silencing increased IL-4 production, the observations suggest that N-Ras is associated with elicitation of TH2 response whereas K-Ras and H-Ras associates with TH1 response. Exactly reverse profile was observed with IFN-γ production. Next we observed that in K-Ras or H-Ras, but not NRas, silenced condition, or under N-Ras overexpression, IL-10 in Foxp3+ -gated cells was higher in Ova-primed BALB/c mice, as compared to naïve. K-Ras or H-Ras overexpression induced significantly fewer T-reg cells. These results suggest that Ras isoforms mediate modulation of Ova-specific T-reg cells. As the Ras isoforms had little influence on T-independent antigen-specific antibody response, it is likely that the Ras isoforms had insignificant influence on these B cells. Given that Ras isoforms had influence on macrophages and also on T cells in a carrier-specific and leishmanial antigen-specific responses, it is most likely that the Ras isoforms played significant roles in macrophages and T cells to regulate antigen-specific immune response.
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5. Concluding remarks Despite the huge structural similarities among the Ras isoforms, the antigen-specific immune responses to the hapten-carrier conjugates and Leishmania major infection are selectively regulated by the Ras isoforms. These novel observations lay the scientific rationale for Ras isoformspecific therapy, which remains to be reported yet.
[7]
[8]
[9]
Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
[10]
[11]
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Contents lists available at ScienceDirect
Cytokine journal homepage: www.elsevier.com/locate/cytokine
Short communication
Ras isoforms selectively regulate antigen-specific immune response ⁎
T
Mukesh Kumar Jha, Aditya Y. Sarode, Bhaskar Saha National Centre for Cell Science, Ganeshkhind, Pune 411007, India
A R T I C LE I N FO
A B S T R A C T
Keywords: Ras isoforms Leishmania TNP-Ova Ras GTPase Cytokine
H-/K-Ras and N-Ras isoforms were proposed to lack functional specificities due to similarity in 1–165 amino acids. As recent studies implied Ras isoform-specific developmental effects, we examined their functional specificity using Leishmania major infection, anti-hapten antibody response and carrier-specific T cell response. While N-Ras overexpression increased L. major infection in resistant C57BL/6 mice, H-Ras or K-Ras overexpression reduced the infection in susceptible BALB/c mice. These Ras isoforms differentially regulated antiTNP antibody response in TNP-Ova-primed, but not in TNP-Ficoll- or TNP-LPS-primed, BALB/c mice. Ras isoform-specific silencing selectively modulated Ova-specific T cell response. The data indicate Ras isoform-specific regulation of antigen-specific immune response.
1. Introduction Antigen-specific immune responses are regulated by many factors including cell signaling intermediates. One of these signaling intermediates is Ras GTPases. Ras GTPases have three major isoforms- NRas, K-Ras and H-Ras. Ras-GTPases were chiefly implicated in oncogenesis and development [1]. Owing to the high sequence homology in their N-terminal 1–165 amino acid residues [2], these ubiquitously expressed Ras isoforms are considered to be functionally redundant [3]. However, an association of K-Ras and N-Ras mutations with pancreatic cancers and leukemia, respectively [4], and indispensable role of K-Ras, but not H-Ras or N-Ras, in mouse development [5] implied non-redundancy among Ras isoforms. These studies focused on the role of Ras in oncogenesis or development but the role of Ras isoforms in modulating antigen-specific immune response is not yet explicitly demonstrated. Therefore, we examined the Ras isoforms’ role in the modulation of antigen-specific immune responses to 2, 4, 6-TrinitrophenylOvalbumin (TNP-Ova), as compared with T-independent antigensTNP-Ficoll and TNP-LPS- as an internal control for antigen-processing and T-dependent immune response. H/-K-Ras overexpression in a susceptible host or N-Ras overexpression in a resistant mouse turns them resistant or susceptible, respectively, to L. major infection. The selective modulation of the anti-TNP antibody response to TNP-Ova by the Ras isoforms matches the profile with the infection indicating Ras isoformspecific regulation of antigen-specific immune response. Anti-TNP
IgG2a production corroborated with IFN-γ production from the ovalbumin-stimulated T-cells in TNP-Ova-primed mice. These data indicate Ras isoform-specific functions paving the way for Ras-targeted therapy against many diseases. 2. Materials and methods 2.1. Reagents Immunization antigens TNP-Ova, TNP-Ficoll, TNP-LPS and TNPBSA (Biosearch Technology, Inc, CA) and EndoFit Ovalbumin (InvivoGen, San Diego, CA). Western blot antibodies for N-Ras, K-Ras, H-Ras, β-actin and Luminol reagent were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-cytokine Abs (IFN-γ, and IL-4) and cytokine standard for ELISA were purchased from BD Biosciences (San Diego, CA). FACS antibodies anti-CD3-PE-Cy7, anti-CD25-APCCy7 and anti-IL-10-FITC (BD Biosciences Pharmingen, San Diego, CA), anti-CD4-PerCP-Cy5.5 and anti-Foxp3-Pacific Blue (BioLegend, San Diego, CA) and anti-CD127-APC (eBioscience, Santa Clara, CA) were purchased. 2.2. Animals, cell line, parasites and infection BALB/c and C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME) were bred in the experimental animal facility at the National Centre for
Abbreviations: TNP-Ova, 2, 4, 6-Trinitrophenyl-Ovalbumin; TNP-Ficoll, 2, 4, 6-Trinitrophenyl-AECM-Ficoll; TNP-LPS, 2, 4, 6-Trinitrophenyl -Lipopolysaccharide; TNP-BSA, 2, 4, 6-Trinitrophenyl-Bovine Serum Albumin; IFA, Incomplete Freund’s adjuvant; IP, Intra peritoneal; SC, Subcutaneous; Ctrl, Control; CV, Control Vector; Inf, Infection ⁎ Corresponding author. E-mail address: [email protected] (B. Saha). https://doi.org/10.1016/j.cyto.2019.154914 Received 2 August 2019; Received in revised form 5 October 2019; Accepted 30 October 2019 1043-4666/ © 2019 Published by Elsevier Ltd.
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maintained by passage through BALB/c mice. Stationary-phase L. major promastigotes (2x106/mouse, s.c. in the hind footpad) were used to infect BALB/c and C57BL/6 mice. Mice were treated s.c in the hind footpad with 5 × 106 transduction units of lentivirus-expressing N-Ras, K-Ras, H-Ras, pGIPZ, pLKO.1 or control empty vector 2 days prior to infection.
Cell Science in Thoren caging units (Thoren Caging System, Hazleton, PA). The progress of infection was monitored weekly by measuring footpad thickness and parasite load was assessed following euthanization five weeks after the infection. All experiments were in accordance with the animal use protocol approved by the Institutional Animal Care and Use Committee and the Committee for the Purpose of Control and Supervision of Experiments on Animals, the regulatory authorities for animal experimentation. P388D1, a macrophage-like cell line, was obtained from ATCC (American Type Culture Collection) and cultured in RPMI-1640 medium (Life Technologies-BRL) containing penicillin (70 µg/ml), streptomycin (100 µg/ml), 2-mercaptoethanol (50 µM), sodium pyruvate (1 µM), HEPES (20 µM) and 10% heat inactivated FCS (Life Technologies-BRL). L. major (strainMHOM/Su73/5ASKH) was maintained in vitro in RPMI 1640 medium containing penicillin (70 µg/ ml), streptomycin (100 µg/ml), 2-mercaptoethanol (50 µM), sodium pyruvate (1 µM), supplemented with 10% FCS and the virulence was
2.3. Peritoneal macrophage collection C57BL/6 female mice were injected with 2 ml thioglycolate (3% i.p.). Four days later, peritoneal exudate cells were harvested in sterile HBSS (Life Technologies-BRL), centrifuged (210g, 8 min at 4 °C), and the pellet was resuspended in RPMI 1640 with 10% FCS. The cells were seeded in 30 mm culture plates and were cultured at 37 °C in a humidified CO2 incubator for 12 h. Non-adherent cells were removed by replacing the medium with fresh RPMI 1640 with 10% FCS. The culture
Fig. 1. Ras isoforms show specificity in modulating Leishmania major infection in BALB/c and C57BL/6 mice and antigen-specific Ras isoforms modulate T-reg numbers. (a. Left panel) Five BALB/c mice/group were treated with overexpressed Ras isoforms or control vector 2 days prior to infection. Two days later BALB/c mice were infected s.c. in hind footpad with 2 × 106 L. major stationary promastigotes. Disease progression was scored weekly by evaluating the net footpad swelling (the thickness of the uninfected left footpad subtracted from the infected right footpad) by digital micrometer (Mitutoyo Japan) for 5 weeks. The Ras isoforms gene silencing was confirmed (insert, 1a). (a. Middle panel) BALB/c mice were sacrificed 5 weeks after the infection, and parasite burden in lymph node cells was assessed. (a. Right panel) Lymph node weight in treated and infected parasite. (b. Left panel) C57BL/6-derived peritoneal macrophages were treated with N-Ras overexpressed or control vector (2 MoI; 48 h), followed by pulsing with purified M10 (10 µg/ml; 6 h). The macrophages were washed and injected s.c. into the hind footpad of C57BL/6 mice (106 macrophages/mouse) weekly for 3 wks. Four weeks later, the mice were challenged with 2 × 106 L. major stationary promastigotes (s.c.) in the hind footpad. Disease progression was scored weekly by evaluating the net footpad swelling (the thickness of the uninfected left footpad subtracted from the infected right footpad) by digital micrometer for 5 weeks. The Ras isoforms gene overexpression was confirmed (insert, 1b). (b. Middle panel) C57BL/6 mice were sacrificed 5 wks after the infection, and parasite burden in lymph node cells was assessed. (b. Right panel) Lymph node weight in treated and infected parasite. The experiments were performed twice and one representative data are shown. Error bars represent mean ± SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. Five BALB/c mice/group were treated with (c) silenced Ras isoform and (d) overexpressed Ras isoforms along with control vector 2 days prior to immunization (s.c with 60 µg TNP-Ova/mouse in IFA) and 21 days after the immunization, splenic T cells were collected. Five mice per group were sacrificed and pooled for FACS staining without any stimulation. T cells were stained with T-reg cell-specific antibodies were acquired on CD3+CD4+CD25+CD127dim gating and analyzed for expression of IL-10 vs Foxp3, as described in Materials and Methods. The values inside graphs represent % of cells. The experiments were performed twice, and one representative data are shown. 2
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2.8. Production of lentiviral particles for Ras isoforms shRNA and control shRNA
was maintained as per the requirements of the experiments. 2.4. Western blotting
A third generation SIN (self-inactivating) lentiviral vector-based system was used. N-Ras construct in pGIPZ vector, H-Ras and K-Ras constructs in pLKO.1 vector and the corresponding scrambled sequence for control constructs were from Open Biosystems (Huntsville, AL). Using a lentiviral packaging system, we generated replication incompetent HIV-1 based lentivirus for all isoform-specific and control constructs. The viral titer was assessed and the virus particles were concentrated according to the manufacturer’s protocol [6].
Cells were washed twice with chilled PBS and lysed in cell lysis buffer (20 mM Tris [pH 7.4], 150 mM NaCl, 1% Nonidet P-40, 10% glycerol, 2 mM EDTA, protease inhibitor mixture [Roche Applied Science, Mannheim, Germany], and phosphatase inhibitor mixture [Pierce, Rockford, IL]). Protein was quantified by a BCA kit (Pierce) or Bradford reagent and an equal amount of protein was run on SDSPAGE. Resolved proteins were blotted onto polyvinylidene difluoride (Millipore, Bedford, MA) and blocked with 5% nonfat dried milk in TBST (25 mM Tris [pH 7.6], 137 mM NaCl, and 0.2% Tween-20). Membranes were incubated with primary Ab at 4 °C overnight, washed with TBST, and incubated with HRP-conjugated secondary Ab. Immunoreactive bands were visualized with the chemiluminescent Luminol reagent (Santa Cruz Biotechnology, Santa Cruz, CA) (6).
2.9. Statistical analyses The significance of difference between the means was calculated from Student’s t-test. The minimum number of mice (female 6–8 weeks old) used for in vivo experiments was five per group. The results were plotted as mean ± standard error of means (SEM) from triplicate sets of experiments. The significance of difference between the means was considered statistically significant when *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.
2.5. FACS-Treg cell analyses T cells from naive and TNP Ova treated mice were studied for Treg cells by multicolour FACS analyses. After blocking with 5% FCS for 30 min at 4 °C, the cells were washed twice with FACS buffer (1XPBS, 10 mM HEPES buffer and 3% FCS) and stained with anti-CD3-PE-Cy7, anti-CD4- PerCP-Cy5.5, anti-CD127-APC and anti-CD25-APC-Cy7 (45 min, 4 °C) and washed twice with FACS buffer. For intracellular staining, the cells were permeabilized using Cytofix/Cytoperm-Plus Kit with GolgiPlug and washed with Perm/Wash buffer. The cells were stained with anti-Foxp3-Pacific Blue, anti-IL-10-FITC antibodies (60 min, 4 °C). Cells were acquired in the CD3+CD4+CD25+CD127dim gate for Treg cells in a FACS CantoII flow cytometer and analyzed for expression of Foxp3 and IL-10 using BD-FACS Diva software (v5.2; BD Biosciences). Cells stained with Isotype-specific antibodies were used as controls.
3. Results 3.1. Ras isoforms overexpression can dictate susceptibility vs resistance in Leishmania infection While Ras isoforms expression and activities in macrophages were differentially regulated by L. major infection [6], higher L. major infection was reported in N-Ras- and H-Ras-deficient L. major-resistant C57BL/6 mice [7]. We overexpressed H-, K-, or N-Ras isoforms and control vector in susceptible BALB/c mice, followed by L. major infection and assessment of the foot-pad thickness (Fig. 1a Left panel), parasite load (Fig. 1a Middle panel), and lymph node weight (Fig. 1a Right panel). We observed that H-/K-Ras overexpression reduced, whereas N-Ras overexpression increased the infection in BALB/c mice (Fig. 1a, left and middle panels) accompanied by draining lymph node hypertrophy (Fig. 1a, right panel). We further overexpressed N-Ras in C57BL/6-derived elicited macrophages that are pulsed with purified L. major MAPK10 (M10) [8]. These macrophages were reintroduced into C57BL/6 mice followed by challenge with L. major. We observed that the recipient L. major-resistant mice were turned susceptible to infection showing significantly higher parasite burden (Fig. 1b). These data indicate that N-Ras overexpression favours L. major infection whereas K-Ras and H-Ras overexpression inhibits L. major infection.
2.6. Sandwich ELISA for quantifying cytokines The culture supernatants from the splenic T cells stimulated with the indicated doses of Ova for 48 h were assayed for IFN-γ or IL-4 by ELISA [6]. Briefly, ELISA plates were coated overnight at 4 °C with purified Abs to IL-4 (1 µg/ml), or IFN-γ (2 µg/ml). Plates were washed thrice (0.05% Tween 20 in 1X PBS) and blocked for 2 h with 1% BSA. Plates were incubated overnight with cytokine standards or culture supernatants. Plates were washed and incubated with respective biotinconjugated detection Abs for 1 h at 25 °C. Plates were washed and incubated with peroxidase-conjugated streptavidin (Roche Applied Science) for 30 min followed by washing and development with tetramethylbenzidine substrate (BD Pharmingen, San Diego, CA). Reaction was stopped by the addition of 1 N H2SO4, and absorbance was measured at 450 nm in an ELISA reader (VERSA max microplate reader, Molecular Devices). The values of standards were plotted and the quantities of the cytokines were estimated in the samples and expressed in pg/mL.
3.2. Ras isoforms-specific regulation of T-reg cells T-regulatory (T-reg) cells were proposed as professional suppressors of immune responses and, in corroboration with that proposition; these cells were implicated in the persistence of Leishmania parasite in a susceptible host [9–11]. As T-reg cells expansion may be dependent on the state of APC activation that, in turn, can be controlled by Ras isoforms, we examined whether silencing of Ras isoforms individually could affect the T-reg cells in Ova-primed BALB/c mice. Therefore, we administered lentivirally-expressed N-Ras shRNA, K-Ras shRNA or HRas shRNA or control shRNA and overexpressed Ras isoforms or control vector into Ova-primed BALB/c mice. The mice were sacrificed 21 days after the priming and the T-reg cells were enumerated by multi-color FACS. We observed higher IL-10 on Foxp3-gated cells in K-Ras and HRas, but not N-Ras (Fig. 1c), silenced condition, whereas Ras isoforms overexpression reversed the phenomena in Ova-primed BALB/c mice (Fig. 1d). These data suggest that Ras isoforms contribute to T-reg cells response.
2.7. Antibody ELISA To detect antigen-specific immunoglobulin, 96 well microtiter plates were coated with 2,4,6-Trinitrophenyl-Bovine Serum Albumin (TNP-BSA-10 µg/ml) at 4 °C (overnight). Using 1% BSA (in 1X PBS), the plates were blocked for 2 h at room temperature and sera (dilutions prepared with the help of 1X PBS) from different groups of mice were added to the coated plates and incubated overnight at 4 °C. The plates were washed four times with wash buffer and bound antibodies were detected using biotin-conjugated goat anti-mouse IgG1, IgG2a and IgM specific antibodies (BD Bioscience; San Diego, CA). Antibody isotypes were measured by ELISA [6]. 3
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Fig. 2. Functional specificities of Ras isoforms in the TNP-Ova primed BALB/c mice. (a). Five female BALB/c mice/group were treated with Ras isoform shRNA Lv or control shRNA Lv 2 days prior to immunization (s.c with 60 µg TNP-Ova, 10 µg TNP-LPS and 50 µg TNP-Ficoll /mouse in IFA) and 10 days after the immunization, sera were collected and checked for the levels of IgG1, IgG2a and IgM antigen-specific immunoglobulin by indirect antibody ELISA method, as described in Materials and Methods. (b) BALB/c mice were treated with overexpressed Ras isoforms or control vector 2 days prior to immunization (s.c with 60 µg TNP-Ova, 10 µg TNP-LPS and 50 µg TNP-Ficoll /mouse in IFA). 10 days after the immunization and checked for the levels of IgG1, IgG2a and IgM antigen-specific immunoglobulin by indirect antibody ELISA in the sera as described in Materials and Methods. Post 21 days of immunization, (c) Ras isoforms silenced or (d) Ras isoforms overexpressed and TNP-Ova immunized mice were sacrificed and collected the splenic T-cells and cultured in presence of the indicated doses of Ova for 48 h. Culture supernatants were assessed for the production of the cytokines IFN-γ and IL-4, as described in Materials and Methods. The experiments were performed thrice, and one representative data are shown. Error bars represent mean ± SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.
3.3. Ras isoforms selectively modulate antigen-specific immune response
4. Discussion
Lentiviral expression of H-Ras or K-Ras shRNA reduced TNP-specific IgG2a antibody whereas N-Ras silencing reduced anti-TNP IgG1 isotype in TNP-Ova primed mice (Fig. 2a and Supplementary Fig. 1a). Ras isoforms overexpression reversed the phenomena (Fig. 2b and Supplementary Fig. 1b). Ova-specific T cell response corroborated with the isotype profile, as H-Ras or K-Ras silencing reduced IFN-γ production whereas N-Ras silencing reduced IL-4 production (Fig. 2c). A reverse profile was obtained in Ras isoforms overexpression (Fig. 2d) indicating the role of Ras isoforms functional specificities in an antigen-specific immune response. Contrary to the effects of TNP-OVA, Ras isoforms silencing or overexpression had little effects on anti-TNP isotypes in response to TNP-LPS (Fig. 2a-b and Supplementary Fig. 2a-b) or TNPFicoll (Fig. 2a-b and Supplementary Fig. 3a-b) immunization. LPS and Ficoll being T-independent antigens, the TNP-specific IgM did not switch to IgG1 or IgG2a. Anti-TNP IgM antibody however switched to IgG1 or IgG2a depending on Ras isoforms manipulated suggesting that Ras isoforms effect on antibody is probably through T cells or antigenpresenting cells. Although Ras isoforms were previously implicated in differential regulation of proliferation, anergy and positive and negative selection of T-cells in thymus [12–15], cellular signaling [16] and in tissue-specific carcinogenesis, the functional specificities are assigned to Ras isoforms by the antigen-specific immune response reported here. Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.cyto.2019.154914.
Our data show how specific Ras isoforms can be targeted for antileishmanial immunotherapy and immunoprophylaxis. The roles played by Ras isoforms in antigen-specific immune response were not previously documented. Involvement of Ras isoforms in CD40 signaling in peritoneal macrophages was indicated [6] but herein, the role of individual Ras isoforms in T cell or B cell response using T- dependent and T-independent hapten-carrier conjugates- TNP-Ova, TNP-Ficoll and TNP-LPS and the role of each Ras isoform in anti-leishmanial immune response have been studied. Both Leishmania infection and the T-dependent hapten-carrier (TNP-Ova) response models show Ras isoformspecific modulation of T cell response. As silencing of N-Ras resulted in reduced carrier-specific T cell’s IL-4 production but K-Ras or H-Ras silencing increased IL-4 production, the observations suggest that N-Ras is associated with elicitation of TH2 response whereas K-Ras and H-Ras associates with TH1 response. Exactly reverse profile was observed with IFN-γ production. Next we observed that in K-Ras or H-Ras, but not NRas, silenced condition, or under N-Ras overexpression, IL-10 in Foxp3+ -gated cells was higher in Ova-primed BALB/c mice, as compared to naïve. K-Ras or H-Ras overexpression induced significantly fewer T-reg cells. These results suggest that Ras isoforms mediate modulation of Ova-specific T-reg cells. As the Ras isoforms had little influence on T-independent antigen-specific antibody response, it is likely that the Ras isoforms had insignificant influence on these B cells. Given that Ras isoforms had influence on macrophages and also on T cells in a carrier-specific and leishmanial antigen-specific responses, it is most likely that the Ras isoforms played significant roles in macrophages and T cells to regulate antigen-specific immune response.
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5. Concluding remarks Despite the huge structural similarities among the Ras isoforms, the antigen-specific immune responses to the hapten-carrier conjugates and Leishmania major infection are selectively regulated by the Ras isoforms. These novel observations lay the scientific rationale for Ras isoformspecific therapy, which remains to be reported yet.
[7]
[8]
[9]
Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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