DataSheet_1_The ST2+ Treg/amphiregulin axis protects from immune-mediated hepatitis.pdf

IntroductionThe alarmin IL-33 has been implicated in the pathology of immune-mediated liver diseases. IL-33 activates regulatory T cells (Tregs) and type 2 innate lymphoid cells (ILC2s) expressing the IL-33 receptor ST2. We have previously shown that endogenous IL-33/ST2 signaling activates ILC2s that aggravate liver injury in murine immune-mediated hepatitis. However, treatment of mice with exogenous IL-33 before induction of hepatitis ameliorated disease severity. Since IL-33 induces expression of amphiregulin (AREG) crucial for Treg function, we investigated the immunoregulatory role of the ST2+ Treg/AREG axis in immune-mediated hepatitis.MethodsC57BL/6, ST2-deficient (Il1rl1-/-) and Areg-/- mice received concanavalin A to induce immune-mediated hepatitis. Foxp3Cre+ x ST2fl/fl mice were pre-treated with IL-33 before induction of immune-mediated hepatitis. Treg function was assessed by adoptive transfer experiments and suppression assays. The effects of AREG and IL-33 on ST2+ Tregs and ILC2s were investigated in vitro. Immune cell phenotype was analyzed by flow cytometry. Results and discussionWe identified IL-33-responsive ST2+ Tregs as an effector Treg subset in the murine liver, which was highly activated in immune-mediated hepatitis. Lack of endogenous IL-33 signaling in Il1rl1-/- mice aggravated disease pathology. This was associated with reduced Treg activation. Adoptive transfer of exogenous IL-33-activated ST2+ Tregs before induction of hepatitis suppressed inflammatory T-cell responses and ameliorated disease pathology. We further showed increased expression of AREG by hepatic ST2+ Tregs and ILC2s in immune-mediated hepatitis. Areg-/- mice developed more severe liver injury, which was associated with enhanced ILC2 activation and less ST2+ Tregs in the inflamed liver. Exogenous AREG suppressed ILC2 cytokine expression and enhanced ST2+ Treg activation in vitro. In addition, Tregs from Areg-/- mice were impaired in their capacity to suppress CD4+ T-cell activation in vitro. Moreover, application of exogenous IL-33 before disease induction did not protect Foxp3Cre+ x ST2fl/fl mice lacking ST2+ Tregs from immune-mediated hepatitis. In summary, we describe an immunoregulatory role of the ST2+ Treg/AREG axis in immune-mediated hepatitis, in which AREG suppresses the activation of hepatic ILC2s while maintaining ST2+ Tregs and reinforcing their immunosuppressive capacity in liver inflammation. </p

DataSheet_1_Substantial heterogeneity of inflammatory cytokine production and its inhibition by a triple cocktail of toll-like receptor blockers in early sepsis.xlsx

IntroductionEarly sepsis is a life-threatening immune dysregulation believed to feature a “cytokine storm” due to activation of pattern recognition receptors by pathogen and danger associated molecular patterns. However, treatments with single toll-like receptor (TLR) blockers have shown no clinical benefit. We speculated that sepsis patients at the time of diagnosis are heterogeneous in relation to their cytokine production and its potential inhibition by a triple cocktail of TLR blockers. Accordingly, we analyzed inflammatory cytokine production in whole blood assays from early sepsis patients and determined the effects of triple TLR-blockade.MethodsWhole blood of 51 intensive care patients sampled within 24h of meeting Sepsis-3 criteria was incubated for 6h without or with specific TLR2, 4, and 7/8 stimuli or suspensions of heat-killed S. aureus or E. coli bacteria as pan-TLR challenges, and also with a combination of monoclonal antibodies against TLR2 and 4 and chloroquine (endosomal TLR inhibition), subsequent to dose optimization. Concentrations of tumor necrosis factor (TNF), Interleukin(IL)-6, IL-8, IL-10, IL-1α and IL-1β were measured (multiplex ELISA) before and after incubation. Samples from 11 sex and age-matched healthy volunteers served as controls and for dose-finding studies.ResultsOnly a fraction of sepsis patient samples revealed ongoing cytokine production ex vivo despite sampling within 24 h of first meeting Sepsis-3 criteria. In dose finding studies, inhibition of TLR2, 4 and endosomal TLRs reliably suppressed cytokine production to specific TLR agonists and added bacteria. However, inflammatory cytokine production ex vivo was only suppressed in the high cytokine producing samples but not in the majority. The suppressive response to TLR-blockade correlated both with intraassay inflammatory cytokine production (r=0.29–0.68; pDiscussionUpon meeting Sepsis-3 criteria for less than 24 h, a mere quarter of patient samples exhibits a strong inflammatory phenotype, as characterized by increased baseline inflammatory cytokine concentrations and a stark TLR-dependent increase upon further ex vivo incubation. Thus, early sepsis patient cohorts as defined by Sepsis-3 criteria are very heterogeneous in regard to inflammation. Accordingly, proper ex vivo assays may be useful in septic individuals before embarking on immunomodulatory treatments.</p

Immune cell depletion during IAV infection in tumour-bearing mice.

Tumours were subcutaneously (s.c.) transplanted and mice were infected with 150 PFU/mL influenza A/PR/8/34 (IAV) as described. 200μg of depleting antibodies against NK1.1 (a), CSF1R (b), CD4 (c) or CD8 (d) were applied i.p. on days 4, 7 and 10 after tumour cell transplantation, respectively. Depletion was confirmed by flow cytometry. Representative dot plots of tumours 12 days after tumour cell transplantation are shown. (TIF)</p

DataSheet_2_Substantial heterogeneity of inflammatory cytokine production and its inhibition by a triple cocktail of toll-like receptor blockers in early sepsis.docx

IntroductionEarly sepsis is a life-threatening immune dysregulation believed to feature a “cytokine storm” due to activation of pattern recognition receptors by pathogen and danger associated molecular patterns. However, treatments with single toll-like receptor (TLR) blockers have shown no clinical benefit. We speculated that sepsis patients at the time of diagnosis are heterogeneous in relation to their cytokine production and its potential inhibition by a triple cocktail of TLR blockers. Accordingly, we analyzed inflammatory cytokine production in whole blood assays from early sepsis patients and determined the effects of triple TLR-blockade.MethodsWhole blood of 51 intensive care patients sampled within 24h of meeting Sepsis-3 criteria was incubated for 6h without or with specific TLR2, 4, and 7/8 stimuli or suspensions of heat-killed S. aureus or E. coli bacteria as pan-TLR challenges, and also with a combination of monoclonal antibodies against TLR2 and 4 and chloroquine (endosomal TLR inhibition), subsequent to dose optimization. Concentrations of tumor necrosis factor (TNF), Interleukin(IL)-6, IL-8, IL-10, IL-1α and IL-1β were measured (multiplex ELISA) before and after incubation. Samples from 11 sex and age-matched healthy volunteers served as controls and for dose-finding studies.ResultsOnly a fraction of sepsis patient samples revealed ongoing cytokine production ex vivo despite sampling within 24 h of first meeting Sepsis-3 criteria. In dose finding studies, inhibition of TLR2, 4 and endosomal TLRs reliably suppressed cytokine production to specific TLR agonists and added bacteria. However, inflammatory cytokine production ex vivo was only suppressed in the high cytokine producing samples but not in the majority. The suppressive response to TLR-blockade correlated both with intraassay inflammatory cytokine production (r=0.29–0.68; pDiscussionUpon meeting Sepsis-3 criteria for less than 24 h, a mere quarter of patient samples exhibits a strong inflammatory phenotype, as characterized by increased baseline inflammatory cytokine concentrations and a stark TLR-dependent increase upon further ex vivo incubation. Thus, early sepsis patient cohorts as defined by Sepsis-3 criteria are very heterogeneous in regard to inflammation. Accordingly, proper ex vivo assays may be useful in septic individuals before embarking on immunomodulatory treatments.</p

IAV infection suppresses tumour growth.

(a-d) 1 x 105 B16F1 (B16) tumour cells were transplanted subcutaneously (s.c.) into the right flank per mouse. 4 days after tumour transplantation, mice were infected intranasally with 150 PFU/mL Influenza A/PR/8/34 (IAV). (a) Viral load was measured by M1 expression relative to RPS9 expression in the lungs of infected mice with or without B16 tumour 8 days post infection (dpi). n = 9 for B16+IAV, n = 6 for IAV (b) Viral load as measured from homogenized lung supernatants by counting plaque forming units (PFU) from plaque assay 8 dpi. (c) Body weight change after IAV infection. Data from 2 experiments with 4–6 mice per group per experiment. (d-g) 1 x 105 B16 (d and g), CT26 (e) or 5 x 105 Lewis Lung Carcinoma (LLC) (f) tumour cells were transplanted s.c. and mice were infected as described in a) with (d-f) IAV or (g) inactivated IAV. The tumour volume was measured everyday once palpable upon infection. d&e: Data from 4 experiments with 3–4 mice per group per experiment, f: n = 4, g: Data from 2 experiments with 4 mice per group per experiment. (h) Viral load as measured by M1 expression relative to RPS9 expression in the tumour or lung of infected mice 12 days post tumour transplantation. (i) B16 tumours were transplanted s.c. with 1 x 105 tumour cells and mice were infected intravenously with Friend Virus (FV) 4 days post tumour transplantation. The tumour volume was scored everyday once palpable. n = 4. Error bars represent SEM. Statistical tests on tumour growth development were performed as Two-way-ANOVA, in Sidak’s multiple comparisons test when two groups were compared or Tukey’s multiple comparisons test when more than two groups were compared. Viral titres were compared with non-parametric t tests. * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001, ns = not significant.</p

Virus-specific CD8⁺ T-cells infiltrate the tumour but are dispensable for tumour growth suppression.

(a) B16 tumour-bearing mice were infected with IAV (150 PFU/mL) as described. 8 days post infection mice were sacrificed for flow cytometry analysis of IAV nucleoprotein (NP)-specific CD8+ T-cells. Frequencies of CD43, CD43 and Granzyme B (GzmB) or Ki67 expressing cells are shown. (b) CD8+ T-cells were isolated from IAV-infected lungs 8 dpi and adoptively transferred (ACT) into B16 tumour-bearing recipient mice at indicated time point. Data from 2 experiments with 4 mice per group per experiment. (c) CD8+ T-cells were isolated from IAV-infected lungs 8 dpi and transferred intra-tumouraly (i.t.) at indicated time point. 4–5 mice per group. Error bars represent SEM. Statistical tests between two groups were performed as unpaired Student’s t-tests. Statistical tests on tumour growth development were performed as Two-way-ANOVA, in Tukey’s multiple comparisons test. (TIF)</p

CD8⁺ T-cells are essential for tumour growth suppression during IAV infection.

Tumours were subcutaneously (s.c.) transplanted and mice were infected with 150 PFU/mL Influenza A/PR/8/34 (IAV) as described. (a) 200 μg anti-IFNAR antibody was applied intraperitoneally (i.p.) on days 4, 7 and 10. n = 6. (b) For Natural Killer (NK)-cell or macrophage depletion 200 μg of antibodies against NK1.1 or CSF1R were applied i.p. respectively on days 4, 7 and 10. n = 3. (c) Rag2-/- or BALB/c wildtype mice were s.c. injected with 1 x 105 CT26 tumour cells followed by IAV (150 PFU/mL) infection. Data from 2 experiments with 3–4 mice per group per experiment. (d) For selective T-cell depletion 200 μg of antibodies against CD4 or CD8 were applied i.p. on days 4, 7 and 10. Data from 2 experiments with 4 mice per group per experiment. Error bars represent SEM. Statistical test on tumour growth development was performed as Two-way-ANOVA followed by Tukey’s multiple comparisons test. ** = p<0.01, **** = p<0.0001.</p

IAV infection reduces activation of Tregs.

B16 tumour-bearing mice were infected with IAV as described. 8 days post infection mice were sacrificed for flow cytometry analysis. (a) Representative dot plots of CD4+ T-cells expressing FoxP3. (b) Frequencies of FoxP3+ of CD4+ T-cells in the tumour. Data from 3 experiments are shown. (c) Mean fluorescent intensity (MFI) of CD25 of FoxP3+ CD4+ T-cells in the tumour. (d) Frequencies of GARP expressing FoxP3+ CD4+ T-cells. Error bars represent SEM. Statistical tests between two groups were performed as Student’s t-tests. * = p<0.05.</p

IAV infection suppresses tumour growth dose dependently.

1 x 105 B16F1 (B16) tumour cells were transplanted subcutaneously (s.c.) into the right flank per mouse. 4 days after tumour transplantation, mice were infected intranasally with Influenza A/PR/8/34 (IAV). The tumour volume was measured everyday once palpable upon infection. Data from 2 experiments with 3–4 mice per group per experiment. Error bars represent SEM. Statistical tests on tumour growth development were performed as Two-way-ANOVA followed by Tukey’s multiple comparisons test. **** = p (TIF)</p

IAV infection impedes differentiation of T-cell exhaustion.

B16F1 (B16) tumour-bearing mice were infected with Influenza A/PR/8/34 (IAV) (150 PFU/mL) as described. 8 days post infection mice were sacrificed for flow cytometry analysis. (a) Frequencies of PD-1 expressing gp100-specific CD8+ T-cells in the tumour. (b) Representative dot plots of CD8+ T-cells expressing PD-1 and TIM-3. (c) Frequencies of PD-1high TIM-3+ or PD-1int TIM-3- of gp100-specific CD8+ T-cells in the tumour. (d) Frequencies of Granzyme B (GzmB) expressing cells of the populations described in c. (e) Frequencies of Thymocyte selection-associated high mobility group box protein (TOX) expressing gp100-specific CD8+ T-cells in the tumour. Data from 2 experiments are shown. Error bars represent SEM. Statistical tests between two groups were performed as Student’s t-tests. * = p<0.05, ** = p<0.01, *** = p<0.001.</p