IL-6 induced by Staphylococcus aureus infection prevents the induction of skin allograft acceptance in mice (V体育官网)

"VSports注册入口" Mice

Female C57BL/6 (B6, H-2b), BALB/c (B/c, H-2d), C3H/HeJ (H-2k) inbred mice, age 8–9 weeks, were purchased from either The Jackson Laboratory (Bar Harbor, ME), The Division of Cancer Treatment at the National Cancer Institute (Frederick, MD), or Charles River (Wilmington, MA). RAG2−/− mice on the C57BL/6 background were purchased from Taconic (Hudson, NY). TCRβδ−/− mice, CD8−/− mice, IL-6−/− mice, and mAct-OVA transgenic mice, all on the C57BL/6 background, were purchased from the Jackson Laboratory. MyD88−/− mice on the BALB/c and C57BL/6 backgrounds were provided by Dr. S. Akira (Osaka University, Osaka, Japan) (19). OTI transgenic mice on a C57BL/6 (CD90. 1 congenic) background whose T cells recognize the OVA257–264 peptide in the context of H-2Kb were a gift from Dr. A. Sperling (University of Chicago, Chicago, IL). OTII transgenic mice on C57BL/6 background (CD45. 1 congenic) whose T cells recognize the OVA323–339 peptide presented by I-Ab were a gift from Dr. Y. X. Fu (University of Chicago, Chicago, IL). TCR75 transgenic mice on a C57BL/6 background whose T cells recognize the H-2Kd-derived peptide (Kd54–68) presented on I-Ab were a kind gift form Dr. P. Bucy (University of Alabama, Birmingham, AL) VSports. Animals were kept in a biohazard facility and used in agreement with the University of Chicago Institutional Animal Care and Use Committee according to the National Institutes of Health guidelines for animal use.

Skin Transplantation

Anti-CD154 mAbs were generated from the anti-CD154 (MR1) hybridoma, which was a generous gift from Dr. K. Bishop (University of Michigan, Ann Arbor, MI). After growth in protein-free hybridoma medium (Invitrogen), anti-CD154 mAbs were purified from hybridoma supernatants using 45% ammonium sulfate precipitation and then dialyzed in PBS for 72 hours VSports app下载. Donor grafts were prepared by harvesting full thickness dorsal flank skin approximately 1 cm in diameter from either wild type BALB/c, MyD88−/− BALB/c, or mAct-OVA transgenic C57BL/6 mice where indicated. Skin grafts were then transplanted onto the dorsal flank of recipient mice that were either wild type C57BL/6 or RAG2−/−, TCRβδ−/−, CD8−/−, MyD88−/−, or IL-6−/− mice all on the C57BL/6 background where indicated. Recipient animals were then treated with anti-CD154 (1mg/dose i. v. on day 0, and i. p. on days 7 and 14 post-transplantation) in combination with DST (107 donor splenocytes on the day of transplantation). Skin grafts were followed visually for rejection, as defined by evidence of graft necrosis or contraction. For IL-6 neutralization studies, purified anti-IL-6 antibody (MP5-20F3, Rat IgG1) was purchased from BioXcell (West Lebanon, NH) and injected post-transplantation (500 μg i. v. , day 0; 250 μg i. p. , days 1, 3, 5, 7, 10, and 14). For IL-17 neutralization studies, purified anti-IL-17 antibody (clone 50104, Rat IgG2a) was purchased from R&D Systems (Minneapolis, MN) and injected post-transplantation (100 μg/mouse i. v. day 0, i. p days 2, 3, 4, 7, 9, 11 and 14). For pharmacologic immunosuppression studies, mice were treated with either methylprednisolone (20 mg/kg, day 0, i. p), cyclosporin (20 mg/kg, day 0, i. p. ), or sirolimus (0. 3 mg/kg, day 0, i. p) purchased from the University of Chicago Hospitals Pharmacy.

V体育官网 - Bacterial Preparations and Mouse Infections

SA strain EsxB:erm, a generous gift from Dr. D. Missiakas (University of Chicago, Chicago, IL) (20), was grown at 37°C overnight in tryptic soy broth (BD Biosciences) containing 10μg/ml erythromycin. PA strain PA01, a generous gift from Dr. J. Alverdy (University of Chicago, Chicago, IL), was grown in tryptic soy broth overnight at 37°C. After overnight growth, SA and LM cultures were diluted 100-fold in fresh media, and incubated at 37°C for 5 hours. LM strain, LM-OVA, was grown in brain-heart infusion broth (BD Biosciences) at 37°C overnight. Bacteria were grown to log phase cultures and titers were quantified by OD₆₀₀. Bacteria were collected by centrifugation, washed twice, and resuspended in PBS. Mice receiving live infections were injected i.p. on the day of transplantation with SA (2×10⁸ cfu/200μl), LM (2×10⁵ cfu/200μl), or PA (2×10⁷ cfu/200μl). Heat killed SA was generated by incubating cultured SA at 60°C for 3 hours. Heat killed SA particles were then washed twice with PBS. Mice receiving heat killed SA were injected with 2×10⁹ cfu/200 μl i.p. on the day of transplantation. Bacterial doses were chosen as the highest sub-lethal dose acceptable after performing an LD₅₀ analysis for each strain.

T cell Proliferation Assays (VSports)

In vivo CD4⁺ and CD8⁺ T cell proliferation was analyzed after adoptive transfer into recipients of skin transplantation. Splenocytes from OTI and OTII mice were processed into single cell suspensions and enriched for CD8⁺ and CD4⁺ T cells, respectively, by negative magnetic separation (Miltenyi Biotec). They were then labeled with carboxyfluorescein succinimidyl ester (CFSE) and adoptively transferred into mice on the day of transplantation (2–5×10⁵ OTI and OTII cells, i.v.). Five days after transplantation splenocytes were harvested, processed into single cell suspensions, and stained for flow cytometry with the following antibodies in 1%BSA/0.02% sodium azide (FACS buffer) for 1 hour at 4°C: 1) OTI staining: PE-labeled anti-TCRVα2 (B20.1, rat IgG2a) was purchased from eBioscience (San Diego, CA). Biotinylated TCRVβ5.1/5.2 (MR9-4, mouse IgG1), PerCP-labeled anti-CD90.1 (OX-7, mouse IgG1), PECy7-labeled anti-CD8 (53–6.7, rat IgG2a), APC-labeled anti-GR1 (RB6-8C5, rat IgG2b), APC-labeled anti-CD19 (1D3, rat IgG2a), APC-labeled anti-CD11b (M1/70, rat IgG2b), APC-labeled anti-CD11c (HL3, hamster IgG1) were purchased from BD Biosciences (Franklin Lakes, NJ). 2) OTII staining: PE-labeled anti-TCRVα2 (B20.1, rat IgG2a), biotinylated anti-CD45.1 (A20, mouse IgG2a) were purchased from eBioscience (San Diego, CA). PECy7-labeled anti-CD4, APC-labeled anti-GR1 (RB6-8C5, rat IgG2b), APC-labeled anti-CD19 (1D3, rat IgG2a), APC-labeled anti-CD11b (M1/70, rat IgG2b), APC-labeled anti-CD11c (HL3, hamster IgG1) were purchased from BD Biosciences (Franklin Lakes, NJ). Secondary staining was performed with APCCy7-labeled streptavidin, purchased from BD Biosciences (Franklin Lakes, NJ). After all staining, cells were washed twice with FACS buffer. Samples were run on the LSR-II (BD Biosciences, Franklin Lakes, NJ) and analyzed for evidence of proliferation as indicated by dilution of CFSE intensity using FlowJo cytometry analysis software (Tree Star, Ashland, OR).

Serum IL-6 Measurements

After C57BL/6 were injected with SA (2×10⁸), LM (2×10⁵), PA (2×10⁵), or IL-6-expressing or control plasmids, blood was collected by retro-orbital puncture at the indicated time points and serum was separated by centrifugation. Serum samples were stored at -20°C for subsequent analysis. Initial serum cytokine screening after SA infection was conducted by Mouse cytokine 20-Plex panel (Invitrogen, Carlesbad, CA). The concentrations of IL-6 were subsequently measured by ELISA (eBioscience, San Diego, CA).

Hydrodynamic IL-6 Gene Delivery

Mouse IL-6 expressing and empty control plasmids were purchased from Invivogen (San Diego, CA). Stock plasmids were isolated and prepared using EndoFree Plasmid Mega Kit (Qiagen, Valencia, CA). Plasmid DNA concentration was determined by spectrometer and confirmed in by agarose gel electrophoresis. Plasmid DNA (5 μg/mouse) was diluted in 1.9ml sterile nonpyrogenic 0.9% sodium chloride solution (Hospira Inc., Lake Forest, IL), and injected through tail vein within 5–6 seconds using a 3ml syringe with 27G_1/2 needle (BD Bioscience, Franklin Lakes, NJ) two days prior to transplantation.

"VSports" IFNγ and IL-17 ELISPOT Assays

The IFN γ and IL-17 ELISPOT assays were conducted according to the instructions of the manufacturer (BD Biosciences, Franklin Lakes, NJ). Briefly, ELISPOT plates (Millipore, Billerica, MA) were coated with purified anti-IFN γ capture antibody or purified anti-IL-17 capture antibody (BD Biosciences, Franklin Lakes, NJ) overnight at 4°C and then blocked with 10%FBS/PBS. Responder splenocytes (10⁶/well, in triplicate) harvested from transplanted mice were co-cultured with γ-irradiated (3000 rads) splenocytes (5×10⁵/well) that were either syngeneic (C57B/L6), allogeneic (BALB/c), third-party (C3H), or syngeneic splenocytes pulsed overnight with heat killed SA (10⁸ cfu/10⁷ splenocytes). Responder cells were also stimulated with or without anti-CD3 (2C11). Cell cultures were incubated for 12 hours at 37°C, 5% CO₂. Biotinylated anti-IFNγ or anti-IL-17 detection antibodies (BD Biosciences, Franklin Lakes, NJ) were added, followed by HRP-conjugated anti-biotin (BD Biosciences, Franklin Lakes, NJ). Plates were developed using 3-amino-9-ethylcarbazole (AEC) substrate. The numbers of spots per well were enumerated using the ImmunoSpot Analyzer (CTL Analyzers LLC, Cleveland).

Statistics

The two-tailed Student’s t test or the Tukey test for multiple comparisons was performed to determine statistical differences between groups.

V体育官网 - SA infection at the time of transplantation prevents the induction of skin allograft acceptance with anti-CD154/DST

We have previously shown that a model intracellular organism, LM, can prevent the induction of skin allograft acceptance (1); however, we were interested in extending those observations and exploring the impact of two clinically important bacterial species, the gram-positive commensal and pathogen, SA, as well as the gram-negative opportunistic pathogen, Pseudomonas aerugninosa (PA). Using a model of skin allograft acceptance induced by anti-CD154/DST, we tested the impact of SA and PA infections on the ability of anti-CD154/DST treatment to induce skin allograft acceptance (Figure 1A). In the absence of anti-CD154/DST treatment, allogeneic skin grafts were rejected within 12 days. Upon treatment with anti-CD154/DST, long-term skin allograft survival was achieved with a median survival of 68 days. Infection with a maximally tolerated dose of PA had no effect on allograft acceptance and the median graft survival was >60 days. Despite administration of anti-CD154/DST, SA infection at the time of transplantation precipitated an acute rejection in 75 percent of recipients of allogeneic skin grafts, with a median graft survival of 15 days. Importantly, SA infection had no effect on survival of syngeneic skin grafts, which persisted long-term indicating that SA did not induce non-specific inflammation that prevented healing of skin grafts, but rather, SA was augmenting allo-specific immune responses.

Rejection associated with SA infection is dependent upon innate immune recognition and signaling

The primary host immune response against SA involves innate immune recognition and activation. SA components can be recognized by TLR2 and TLR4, and these TLRs together with downstream MyD88-signaling have been reported to be critically important to host-defense against SA infection (21–24). We tested whether these early innate events contribute to SA-mediated prevention of graft acceptance by using MyD88^−/− donor-recipient pairs. Because MyD88^−/− mice are exquisitely sensitive to SA infection (23, 25) heat-killed bacteria were administered in lieu of live infection to avoid mortality. Similar to live SA infection, heat-killed SA were capable of preventing graft acceptance resulting in acute skin allograft rejection in wild type animals (Figure 1B). Acute skin allograft rejection with normal kinetics was observed in MyD88^−/− mice in the absence of anti-CD154/DST treatment; however, SA was unable to precipitate acute rejection in anti-CD154/DST-treated MyD88^−/− recipients (Figure 1B). These data indicate that innate recognition of SA and events downstream of MyD88-signalling are necessary for the ability of SA to prevent graft acceptance.

Rejection associated with SA infection is a classical, T cell-mediated response

Allograft rejection is considered a T cell-dependent phenomenon that can be shaped by stimuli that active innate immunity (26). A role of T cells, especially Th17 cells, in controlling acute SA infection has recently been revealed (27, 28). We tested whether the adaptive immune system was required for SA-mediated prevention of skin allograft acceptance or whether innate immunity was sufficient to mediate rejection. Using RAG^−/− recipients, we observed that an innate immune activation alone was not sufficient for SA to mediate rejection and that lymphocytes were required. Using TCRβδ^−/−, which lack both conventional αβ T cells as well as Γδ T cells, we also observed that T cells were required for SA-induced prevention of tolerance. Specifically, we demonstrate by using CD8^−/− recipients, that CD8⁺ T cells were necessary for SA-mediated allograft rejection (Figure 1C). We could not specifically address the question of whether CD4⁺ T cells were required in this model of SA-mediated prevention of skin allograft tolerance, as tolerance could not be established in their absence (29). These data led us to conclude that, although innate immune responses elicited by SA are important for host defense against the bacterium, they were, in and of themselves, not sufficient to induce rejection in anti-CD154/DST-treated recipients. Rather, our data suggest that SA infection over-rides the effects of anti-CD154/DST to facilitate a T cell-dependent rejection process.

SA promotes the CD154-independent expansion of alloreactive CD4⁺ and CD8⁺ T cells

We sought to visualize the direct effects of infection on the fate of alloreactive T cell populations by using an adoptive transfer system in which OVA-specific CD4⁺ OTII and CD8⁺ OTI TCR transgenic cells were CFSE labeled and transferred into C57BL/6 recipients of mOVA.B6 skin grafts. Animals were sacrificed 5 days after transfer and transplantation for flow cytometric analysis of the transgenic populations within the spleen (Figure 2). We observed that in the absence of transplantation, OTII and OTI T cells remained largely undivided either in the absence or presence of SA. Upon transplantation and in the absence of anti-CD154/DST treatment, OTII and OTI T cells proliferated vigorously. This proliferation was largely suppressed with anti-CD154/DST treatment. In contrast, in the setting of concomitant transplantation and SA infection, proliferation of both CD4⁺ (OTII) and CD8⁺ (OTI) alloreactive T cells was observed in anti-CD154/DST-treated recipients.

SA-induced IL-6 is necessary and sufficient for prevention of skin allograft acceptance

We hypothesized that proinflammatory cytokines induced downstream of MyD88 following SA infection may impact T cell alloreactivity and confer resistance to anti-CD154/DST treatment. Experiments using a mouse cytokine 20-Plex panel (Invitrogen) quantified the levels of 20 cytokines and chemokines in the serum 1–24 hours after SA infection. IL-6 was observed to be a cytokine that was strongly induced early following SA infection, and moderately increased in LM-infected animals, but not following PA infection (Figure 3A). To test whether IL-6 was important for the ability of SA to prevent graft acceptance, we used IL-6^−/− recipients as well as anti-IL-6 neutralization in wild-type recipients treated with anti-CD154/DST. We observed that infection with SA did not result in the rejection of skin allografts in the absence of IL-6, and conclude that IL-6 is critical for the ability of SA to prevent skin allograft acceptance (Figure 3B). Normal rejection kinetics was observed in IL-6^−/− recipients that were not treated with anti-CD154/DST. In addition, LM infection, with mediates IFNs-dependent allograft rejection (13), was able to precipitate acute rejection in IL-6^−/− recipient animals treated with anti-CD154/DST (Figure 3B).

We next sought to determine whether IL-6 alone, in the absence of infection with SA, was sufficient to prevent graft acceptance. To this end, we used the system of hydrodynamic gene delivery (30–33) to achieve levels of systemic IL-6 comparable to those seen after SA infection (Figure 4A). The delivery of the IL-6 expressing plasmid, but not the empty control plasmid, was able to prevent induction of allograft acceptance (Figure 4B). From these data, we infer that IL-6 produced during inflammation or upon bacterial infection is sufficient to promote activation of alloreactive T cells and the acute rejection of the allograft in anti-CD154/DST treated recipients.

VSports app下载 - SA and IL-6 promote the differentiation of an alloreactive Th1, but not alloreactive Th17, responses

SA-induced IL-6 has the potential to influence the activity of alloreactive T cells in a number of ways. IL-6 is known to promote the proliferation of Th1 effector cells, thereby leading to their escape from regulation (34). In addition, IL-6 has recently been described to be a pivotal cytokine in driving the differentiation of Th17 effector cells at the expense of peripheral Treg induction (35). Because this model of costimulation blockade-mediated allograft acceptance with anti-CD154/DST is thought to be dependent upon a balance of effector and regulatory T cells (6), we characterized the impact of IL-6 on this equilibrium. To this end, we used a 24-hour IFNγ or IL-17 ELISPOT assay with recipient splenocytes as responders and irradiated splenocytes that were syngeneic, allogeneic or syngeneic pulsed with heat-killed SA as stimulators. In the absence of anti-CD154/DST treatment, we observed that the donor-stimulated response was primarily associated with the presence of IFNγ-producing cells. These cells were largely absent in the setting of graft acceptance induced by anti-CD154/DST. In the setting of a SA infection or exogenous IL-6 treatment, there was a partial restoration of this primed IFNγ-producing population of alloreactive cells (Figure 5A). Following SA infection, there was a detectable population of primed, IL-17-producing anti-SA cells, however there were no detectable IL-17-producing alloreactive cells upon SA- or IL-6-mediated rejection (Figure 5B). These observations suggested that IL-6 enhanced conventional Th1 alloreactive responses but had minimal affect in skewing to a Th17 response in the setting of allograft rejection. This conclusion was further substantiated by the inability of neutralizing anti-IL-17A mAbs to restore graft acceptance to recipients treated with anti-CD154/DST and infected with HKSA (Figure 5C). This dose of anti-17A mAb had previously been shown to prevent CpG-mediated cardiac allograft rejection in IL-6-KO mice treated with anti-CD154/DST (36). HKSA was used to ameliorate potential defects in clearing SA in the absence of IL-17, and has been shown to be able to prevent graft acceptance in anti-CD154/DST-treated recipients (Fig 1B).

Treatment with conventional immunosuppression blunts IL-6 production following SA infection allowing for the establishment of graft acceptance despite infection

Steroids have been a cornerstone of immunosuppressive regimens and are known to potently suppress the production of a number of proinflammatory cytokines (2, 37). We hypothesize that inclusion of low-dose steroids to tolerance inducing regimens may modulate the production of inflammatory cytokines after SA infection, thereby allowing for the establishment of allograft acceptance. Consistent with previous reports, we showed that in a naïve animal, a single treatment on the day of transplantation with methylprednisolone (20 mg/kg, i.p), but not the T cell directed therapies of cyclosporine (20 mg/kg, i.p.) or sirolimus (0.3 mg/kg, i.p), blunted the induction of IL-6 following SA infection (Figure 6A). Importantly, upon addition of steroids, we observed that SA infection at the time of transplantation no longer precipitated graft rejection and skin allografts were maintained long-term (Figure 6B).