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Heart failure (HF) is a leading cause of mortality. Inflammation is implicated in HF, yet clinical trials targeting pro-inflammatory cytokines in HF were unsuccessful, possibly due to redundant functions of individual cytokines. Searching for better cardiac inflammation targets, here we link T cells with HF development in a mouse model of pathological cardiac hypertrophy and in human HF patients. T cell costimulation blockade, through FDA-approved rheumatoid arthritis drug abatacept, leads to highly significant delay in progression and decreased severity of cardiac dysfunction in the mouse HF model. The therapeutic effect occurs via inhibition of activation and cardiac infiltration of T cells and macrophages, leading to reduced cardiomyocyte death. Abatacept treatment also induces production of anti-inflammatory cytokine interleukin-10 (IL-10). IL-10-deficient mice are refractive to treatment, while protection could be rescued by transfer of IL-10-sufficient B cells. These results suggest that T cell costimulation blockade might be therapeutically exploited to treat HF.
Heart failure (HF) is a major cause of hospitalization, morbidity and mortality; it is often encountered as the final stage of pathological cardiac hypertrophy and fibrosis brought about by hemodynamic overload1. Some forms of cardiomyopathy—termed inflammatory cardiomyopathies—are caused by autoimmunity or by immune responses to infection, indicating that cardiac dysfunction can also result from disease of the immune system2. Intriguingly, recent studies have uncovered that HF induced by hemodynamic overload also involves a significant inflammatory component3,4,5. This inflammation is characterized by the presence of innate immune cells (macrophages) in the myocardium and upregulation of pro-inflammatory cytokines, such as tumour-necrosis factor-α, interleukin (IL)-6 and IL-1β, which impact negatively on disease outcome3,6,7. Even though its absence can be compensated8, IL-6 administration is sufficient to set off the process leading to pathological cardiac hypertrophy9. Innate immune cells and cytokines are believed to promote cardiac inflammation, worsening disease outcome.
Although the concept of inflammation as a major component of HF is consolidated10, clinical trials attempting to combat HF by blocking cytokines have not been successful5,11. The reason for this failure could be the redundant function of individual cytokines8. Therefore, in order to identify more suitable immunotherapy targets for HF, we need to better characterize the involvement and hierarchy of different soluble and cellular (innate and adaptive) immune mediators in the disease.
The innate immune system acts as a non-specific, but effective and rapid, first line of defense against pathogens. During long-lasting responses, however, it becomes subject to the control of the adaptive immune system's T lymphocytes (T cells)12, which, along with B cells, mediate antigen-specific immune responses. Therefore, T cells, if involved in HF pathogenesis, could become attractive and more specific immunotargets for therapeutic intervention. This assumption is supported by the implication of T cells in pressure overload-induced cardiac fibrosis13.
Here we identified the immune mediators involved in pressure overload-induced HF, finding that T cells infiltrated the pathologically hypertrophic myocardium, in line with their role in long-lasting inflammation. Indeed, inflammation was a key factor distinguishing pathological hypertrophy from physiological, 'benign' hypertrophy, which occurs during exercise training. Taking advantage of the presence of T cells, we utilized abatacept—an Food and Drug Administration (FDA)-approved CTLA4-Ig fusion protein that blocks T cell costimulation, selectively inhibiting pro-inflammatory T cell function14—to significantly blunt cardiac dysfunction in a mouse HF model. Inhibition of disease progression was achieved even when the drug was administered at an advanced stage of the pathology. Abatacept systemically inhibited T cell activation, cardiac macrophage maturation and reduced cardiac T cell and macrophage infiltration, leading to reduced cardiomyocyte death. The protective effect was lost in the absence of anti-inflammatory cytokine interleukin-10 (IL-10), which was produced mostly by B cells. Adoptive transfer of IL-10-sufficient B cells but not T cells into IL-10-deficient recipient mice in the HF model rescued the loss of protection. Taken together, our findings indicate that T cell-mediated responses are involved in the development of pathological cardiac hypertrophy and that interfering with these responses, using existing, clinically validated strategies, has the potential to become a therapeutic option for HF.
We subjected mice to transverse aortic constriction (TAC), the standard model for pathological cardiac hypertrophy15, and assessed the presence of soluble and cellular immune mediators within the myocardium via quantitative PCR (qPCR) at 1 and 4 weeks after TAC surgery (Fig. 1). Cardiac functionality was monitored via regular transthoracic echocardiography (Supplementary Table 1). At 1 week post-TAC, we found a significant upregulation of Tnfa and Il6, as previously described7,16. Cells of the immune system are recruited to and/or retained at their sites of action via chemokines. We found a significant early expression of Ccl2 and Cxcl11 (ref. 17) as well as Ccl4, Ccl5 and Cxcl10 (Fig. 1), the majority of which are markers of a type 1 (M1/Th1)-polarized inflammatory response18. Itgam (CD11b), a hallmark of the presence of innate immune cells, such as macrophages or monocytes, was also upregulated 1 week post-TAC, suggesting that type 1-polarized innate immune cells are recruited to the stressed myocardium early on.
We observed significant upregulation of the T-cell-specific marker Cd3e at 4 weeks post-operation, suggesting that T cells expand or are recruited to the stressed left ventricle at this later timepoint. Concurrent upregulation of Il4, a hallmark of type 2 (M2/Th2)-polarized responses, is compatible with a gradual shift from an M1 to an M2/Th2 response as the myocardium progresses toward HF, though this is speculative. Th2-polarized T cells promote fibrosis in other pathological conditions19. Transcripts of cytokines that characterize Th1 and Th17 responses, such as Ifng and Il17, or of the anti-inflammatory cytokine Il10 were not significantly altered (Supplementary Fig. 1a).
We asked whether the onset of inflammation correlated with T cell infiltration and/or proliferation. Assuming a linear regression model, we first examined the correlation between Cd3e expression (indicative of T cell presence) and Il6 expression (indicative of inflammation) in samples derived from TAC-operated mice, 4 weeks post-operation. The results (Supplementary Fig. 1b, red line) show a significant positive slope, suggesting that such a correlation exists. A likely interpretation would be that inflammation drives the infiltration and/or proliferation of T cells into the myocardium. Repeating the analysis for sham-operated animals (Supplementary Fig. 1b, blue line) also yielded a significant positive slope, however with lower mean il6 and cd3e values. This suggests that, even in the absence of the aortic constriction, the limited (but nonetheless present) inflammation generated by the sham operation (which does involve surgery, albeit without permanent constriction) may be leading to a limited infiltration/proliferation of T cells, even if this is significantly lower than in TAC (as shown in Fig. 1).
The above show that pathological cardiac hypertrophy, which leads to fibrosis and HF, is associated with inflammation. Yet non-pathological forms of cardiac hypertrophy also exist. The most physiologically relevant model for these is exercise training. Mice subjected to a running program show 'physiological' hypertrophy in which the increase in cardiomyocyte size is accompanied by an increased functionality of the cells and absence of fibrosis20,21. We thus asked whether the immune mediators that we identified in the TAC model of HF were also present in exercise-trained mice. We found no significant upregulation of immune response mediator transcripts in these mice (Supplementary Fig. 2a). This finding strongly suggests that, unlike pathological hypertrophy, physiological hypertrophy features a complete absence not only of fibrosis, but also of an innate and adaptive immune response. A more 'artificial', non-pathological hypertrophy model, induced by cardiac-specific overexpression of the constitutively active E40K mutant of the serine-threonine kinase Akt in the heart22, displayed an incomplete array of pro-inflammatory mediators present in the left ventricle of 8-week-old Akt transgenic mice (Supplementary Fig. 2b). Altogether, these results support a positive association between inflammation and the pathological nature of cardiac hypertrophy.
Inhibition of inflammation as a strategy against HF has been attempted before, but the targets utilized resulted to be inadequate for this end5,11. T cells are required for the maintenance of long-term immune responses12 and thus could represent a better therapeutic target. Driven by the finding of T cell-specific Cd3e messenger RNA (mRNA) upregulation in TAC mice at 4 weeks post-TAC, we further investigated the presence of T cells in pathological hypertrophy. Examining mouse left ventricles by immunohistochemistry with anti-CD3e (Fig. 2a), we found that T cells were significantly more abundant in TAC versus sham mice at 4 weeks (Fig. 2b), confirming the mRNA data. T cells react in an antigen-specific manner, involving few specific clones that subsequently expand in number. Thus, we hypothesized that T cells should also be detectable in the heart at an early stage of disease. We thus performed immunohistochemistry analysis on mice at 1 week post-TAC, and indeed we were able to detect T cells (Fig. 2c). We also performed lymphocyte-enriching gradient purification on cardiac suspensions from hearts of mice at 1 week post-TAC, and detected CD3e-expressing cells in the resultant cell populations by flow cytometry (Fig. 2d). Therefore, T cells were present in the hypertrophic myocardium even at an early stage of the pathology.
Studies in the TAC model have identified that cardiac dysfunction can be detected as early as 2 days post-TAC7. T cell activation is often initiated at the lymph nodes that drain the site of inflammation. We thus examined via flow cytometry whether, at 2 days post-TAC, T cells were activated in the heart-draining (mediastinal) lymph nodes. We also examined non-draining (inguinal) lymph nodes as well as spleens of the same animals. At day 2, a significant upregulation of the activation marker CD25 could be seen among CD3+ T cells in the heart-draining lymph nodes, though not in the more distal, non-draining lymphoid compartments (Fig. 2e; gating strategy shown in Supplementary Fig. 3a). T cell presence in the ailing myocardium could create an opportunity to manipulate their function for therapeutic purposes.
In order to confirm the relevance of our findings for human disease, we examined T cell abundance in cardiac tissue derived from HF patients suffering from primary cardiomyopathy. We examined tissue from patients carrying lamin A/C mutations, which, as previously described23, lead to dilated cardiomyopathy and HF. A subset of these carried a second mutation in titin, leading to a more severe dilated cardiomyopathy. We chose these patients as their cardiomyopathy is caused by a non-immunological cause, unlike inflammatory, autoimmune or viral cardiomyopathies2. Detection of T cells in the left ventricle of these patients would suggest that presence of T cells is correlated not only with cardiomyopathies initiated by excessive immune responses, but also with cardiomyopathies triggered by non-immune causes. Cardiac samples were obtained during left ventricular assist device (LVAD) placement surgery, attesting to the advanced stage of their cardiac dysfunction23. Azan's trichrome collagen staining (Fig. 2f) confirmed presence of fibrosis in these specimens (Fig. 2g). Analysis of T cell abundance via CD3e immunohistochemistry (Fig. 2h) in the same samples revealed the presence of infiltrating T cells (Fig. 2i), similar to hearts of mice at 4 weeks post-TAC. In addition to the above, we also examined samples from patients suffering from aortic stenosis, which leads to HF24 and represents the clinical condition that is mechanistically closest to the TAC mouse model. Left ventricles from patients with this form of cardiomyopathy also demonstrated a similarly increased fibrosis (Fig. 2j) and T cell presence (Fig. 2k). Taken together, while only associative, these results further support a link between T cell presence, cardiac fibrosis and pathological hypertrophy.
We hypothesized that specific inhibition of T cell function would have a beneficial effect on HF. CTLA4 is one of the inhibitory molecules through which naturally occurring regulatory T cells, as well as pro-inflammatory T cells at the termination of a response, suppress T cell activation under physiological conditions25. It blocks the CD80/CD86 costimulation signals that T cells must receive from antigen presenting cells (dendritic cells, B cells or macrophages) in order to become fully activated14. CTLA4-Ig fusion protein (abatacept, an FDA-approved drug for rheumatoid arthritis, an autoimmune disease) is a stable, soluble form of CTLA4. We, therefore, tested whether administration of abatacept produced beneficial effects in the TAC model of HF. We treated mice that had been TAC- or sham-operated with three intraperitoneal injections per week of 200μg of abatacept, for 4 weeks, starting 2 days after the operation. As controls, TAC- and sham-operated mice received PBS, at the same timepoints. Cardiac function was monitored by transthoracic echocardiography (see Supplementary Table 2). Day 2 post-operation was chosen as the first timepoint of treatment as significant cardiac dysfunction (increase in left ventricle thickness) can already be detected at 2 days post-TAC via clinically-relevant diagnostic techniques (echocardiography)7.
PBS-treated TAC-operated mice at 1 and 4 weeks post-operation displayed a significant reduction in cardiac function, expressed as percent fractional shortening (FS) or ejection fraction (EF) compared with sham controls, while abatacept-treated mice had no significant difference in FS or EF from sham controls (Fig. 3a,b). Difference in FS was evident from the first week post-TAC operation, up to the end of the experiment (Fig. 3a); the difference in EF increased in significance with time between the PBS- and abatacept-treated groups (Fig. 3b). Hence by administering abatacept starting from 2 days after TAC surgery, we were able to significantly reduce the extent and delay the progression of degradation of cardiac function. The beneficial effect of abatacept was also evident by analysing other hemodynamic parameters, including end-diastolic and end-systolic left ventricular internal diameter (LVIDd and LVIDs) (Fig. 3c,d). Other measured parameters are reported in (Supplementary Table 2). It should be noted that at 3 weeks post-operation, a transient yet significant difference between abatacept-treated and sham control animals could be seen. At the end of the fourth week, we assessed the morphometric indicators of cardiac hypertrophy: heart weight to body weight ratio (Supplementary Fig. 3b), left ventricle to body weight ratio (Supplementary Fig. 3c), heart weight to tibia length ratio (Supplementary Fig. 3d; representative images in Fig. 3e). Abatacept-treated TAC-operated mice displayed significantly lower hypertrophy than PBS-treated controls, according to most of these parameters. Analysis of myocardial 'stress genes', hallmarks of cardiac hypertrophy and failure, in the left ventricles by qPCR also showed a significant upregulation of β-Myosin heavy chain (Mhy7) (Supplementary Fig. 3e), Brain Natriuretic Peptide (Nppb) (Supplementary Fig. 3f) and Atrial Natriuretic Factor (Nppa) (Supplementary Fig. 3g) mRNAs for the PBS- but not for the abatacept-treated groups. Thus, abatacept treatment significantly reduces the severity and delays the progression of the cardiac dysfunction caused by the ventricular pressure overload.
We examined sections with Azan's trichrome staining in order to assess the levels of fibrosis26. A comparison of collagen intensity in identical regions sampled for all treatment groups identified significant increases in fibrosis levels for all TAC-operated groups except for the mice treated with abatacept (Fig. 3f). These results suggest that the beneficial effect of abatacept is also reflected in protection from cardiac fibrosis, a biological response invariably linked to HF27.
Abatacept is based on human CTLA-4 fused with human immunogloblin, but it has been extensively shown to function in mice, due to the high similarity of human and mouse CTLA-4 (refs 28, 29). As human Ig administration could be immunogenic in mice, we included a further set of non-operated mice that received abatacept or an isotype control immunoglobulin (Ig), to assess any effects of the human Ig used in the fusion protein. Neither abatacept alone nor human IgG control injections led to any significant effects in heart function in non-operated animals (Supplementary Fig. 4a,b), suggesting that any alloreactivity to the immunoglobulin had limited effects. Nonetheless, the potential for alloreactivity of the IgG control, in the absence of the immunosuppressive CTLA-4 domain, could possibly worsen the TAC-induced inflammation. For this reason, we chose to use PBS administration rather than IgG administration as a control for our experiments, so as to avoid any deleterious effect on the controls creating the appearance of a stronger therapeutic effect in the abatacept-treated group. Indeed, when we assessed the in vivo effect of abatacept in TAC-operated mice, we found that its protective effect appeared to be even more significant when compared with isotype control-treated rather than PBS-treated TAC-operated mice (Supplementary Fig. 4c–f). This confirmed the validity of our choice of controls.
We next wondered whether abatacept treatment would be able to block the progression of cardiac dysfunction if administered only at a late timepoint, when the disease is more advanced. We thus repeated the in vivo treatment with abatacept, albeit commencing the first treatment at 2 weeks post-TAC, instead of 2 days post-TAC. As it can be seen (Fig. 3g,h) treatment at a late timepoint was able to significantly block further reduction of FS and EF in treated animals. A significant protective effect was also observed in LVIDs, though not LVIDd (Supplementary Fig. 4g,h). These results demonstrate that even late treatment with the drug may have substantial beneficial effects in limiting the progression of HF.
Extensive studies have shown that CTLA4-Ig inhibits T cell function by blocking the costimulatory receptors on antigen presenting cells, which are required for the full activation of pro-inflammatory T cells14,30. The CTLA-4 molecule represents one of the main available mechanisms through which already initiated T cell responses can be physiologically downregulated31,32. Indeed, we found that in vitro abatacept administration to splenocytes inhibited T cell responses (Supplementary Fig. 5a). We, therefore, sought to dissect how abatacept was affecting T cell activation in pathological cardiac hypertrophy. For this, we examined via flow cytometry the expression of activation marker CD25 in T cells at an early timepoint (1 week post-TAC), which is likely to be within the relevant time window for activation events. Abatacept significantly reduced the percentage of CD25+ cells among T cells, not only in the heart-draining (mediastinal) lymph nodes, but also in inguinal lymph nodes and spleen (Fig. 4a). This suggests that abatacept exerted a systemic dampening of T cell activation. CD25 expression on the T cells infiltrating the heart could not be reliably assessed due to the low number of T cells found in the heart at 1 week post-TAC, which renders flow cytometric analysis of subpopulations technically challenging. Reduced T cell activation is likely to lead to reduced proliferation and lower T cell numbers at later timepoints. Indeed, at 4 weeks after surgery, the myocardium of abatacept-treated mice displayed significantly fewer infiltrating T cells than PBS-treated mice (Fig. 4b).
Abatacept has also been shown to inhibit T cell-dependent monocyte/macrophage activation and function33 and B-cell function34, as these cells physiologically provide costimulation to T cells via CD80/CD86. We thus wondered whether abatacept administration in TAC-operated animals led to inhibitory effects on macrophage activation, which has been shown to contribute to cardiac pathology35. We assessed via immunohistochemistry the expression of AIF-1 (Iba-1), a marker of T cell-derived macrophage activation36,37, in the hearts of operated mice, at 1 week post-surgery. In TAC-operated mice, abatacept treatment led to a significant reduction in AIF-1 signal compared to PBS-treated controls (Fig. 4c). Sham-operated mice had negligible signals of AIF-1+ cells (Supplementary Fig. 5b). At 4 weeks post-surgery, the difference in AIF-1+ macrophages between the TAC-operated groups was minimal (Supplementary Fig. 5c), most likely as the overall levels of AIF-1+ macrophages, or indeed total CD11b+ innate immune cells (Fig. 1) in TAC-operated mice is reduced at this late stage of the pathology.
We next examined the maturation state of macrophages38 in the left ventricles of abatacept or control-treated TAC mice at 1 week post-operation, by flow cytometric analysis. We considered the percentage of Ly6C+F4-80+ (immature macrophages) or Ly6C-F4-80+ (mature macrophages) out of CD11b+CD45+ live single cells (gating strategy shown in Supplementary Fig. 5d). We found that hearts of abatacept-treated animals had significantly higher percentage of immature macrophages (Fig. 4d) and significantly lower percentage of mature macrophages (Fig. 4e), compared with controls.
The above findings suggest that abatacept inhibits T cell activation and infiltration/proliferation, but also targets the activation and maturation state of macrophages in the myocardium.
The effect of abatacept on T cell activation occurs via the removal of pro-inflammatory, costimulatory signals32 on antigen presenting cells39. Yet it can additionally be dependent on the production of anti-inflammatory signals, actively inhibiting the pathogenic response30,40. To investigate this, we examined the presence of immune mediators via real-time qPCR in the left ventricles of treated TAC-operated animals. At 1 week post-operation, a timepoint when abatacept already leads to cardioprotective effects, mRNA expression for the pro-inflammatory cytokine IL-6 was significantly upregulated in both abatacept- and PBS-treated TAC-operated mice (Fig. 4f: il6). However, only in abatacept-treated mice could we observe a significant upregulation of mRNA for the cytokine IL-10 (Fig. 4f: il10). IL-10 is one of the most potent anti-inflammatory cytokines utilized by the immune system to shut down unwanted or no-longer-needed responses and it has been shown to mediate cardio-protective effects in HF41, its effect on cardiomyocyte function being opposite to that of IL-6 (ref. 9). Direct in vitro administration of abatacept on cultured neonatal cardiomyocytes did not have any effects on their hypertrophic state (Supplementary Fig. 6a). These findings collectively suggest that abatacept could be mediating anti-inflammatory and subsequent anti-hypertrophic effects via the action of IL-10. As Il10 was upregulated in abatacept-treated TAC mice, we assessed which subset of immune cells could function as IL-10 sources. We examined the expression of intracellular IL-10 by flow cytometry in splenocytes exposed in vitro to abatacept. We found that abatacept induced IL-10 mostly on antigen-presenting cells, the vast majority of which were B cells, while a few IL-10 producing T cells could also be identified (Supplementary Fig. 6b,c).
We thus examined whether IL-10 was necessary for the protective effects of abatacept. We analysed the effect of abatacept on mice deficient for IL-10 (Il10 KO) subjected to TAC. The hallmark of abatacept function is the suppression of T cell responses14,30. Interestingly, in Il10 KO TAC-operated mice, abatacept could no longer inhibit T cell presence in the heart (Fig. 5a), demonstrating that IL-10 is required for the T cell-attenuating, anti-inflammatory effect of the drug. Subsequently, we asked whether IL-10 was necessary for the abatacept-mediated effects on cardiac hypertrophy. Echocardiographic analysis of TAC-operated, Il10 KO mice confirmed that IL-10 was required for the beneficial effect of abatacept on the heart (Fig. 5b–e). Finally, apoptosis of cardiomyocytes is a hallmark of pathological hypertrophy26. While abatacept significantly reduced the extent of cardiomyocyte apoptosis in wild-type TAC-operated mice, this did not occur in Il10 KO mice, which were refractive to treatment (Fig. 5f).
We thus sought to confirm whether the IL-10 producing cells identified above (that is, mostly B cells, and—to a lesser extent—T cells) could be sufficient to rescue the loss of the protective effect in Il10 KO animals. To achieve this, we first transferred 2 × 106 wild-type (Il10-sufficient) B cells or 2 × 106 wild-type (Il10-sufficient) T cells into Il10 KO recipients. We then performed TAC surgery followed by abatacept or control treatment, starting from day 2 post-operation. Transfer of Il10 wild-type B cells was sufficient to rescue the loss of the abatacept-mediated protective effect in Il10 KO TAC-operated mice (Fig. 5g,h: closed squares). On the other hand, transfer of Il10 wild-type T cells could not rescue the protective effect (Fig. 5g,h: open squares). From this we conclude that IL-10 produced by B cells in response to abatacept must be involved in the mechanism of the abatacept-mediated cardioprotective effect. To assess whether this B cell-mediated effect was dependent on the drug's effect on T cells or whether it could be a direct effect on B cells, we assessed the capacity of splenocytes to produce IL-10 after abatacept administration in vitro, in the presence or absence of T cells. We found that the production of IL-10 was unaffected by the absence of T cells (Supplementary Fig. 6d,e), suggesting that the B cell-mediated effect may be direct.
Our results, taken together, suggest that abatacept may protect against the progression of HF by inhibiting the pathogenic immune response mediated by T cells and macrophages, while also directly inducing the beneficial production of anti-inflammatory cytokine IL-10 by B cells.
In this report we demonstrate how abatacept, an FDA-approved drug that inhibits T cell costimulation, reduces severity and delays progression of pressure overload-induced cardiac hypertrophy and fibrosis. Importantly, we were able to demonstrate that the drug could significantly limit the progression of pathology even when administration commenced at a late stage of disease. This was possible because HF pathogenesis is associated with an innate and adaptive immune response. Abatacept blunted this response, and hence inhibited cardiac pathology, via a mechanism dependent on IL-10.
The cardiac inflammation associated with HF is triggered by pro-inflammatory cytokine secretion by stressed cardiomyocytes3,6,7. These cytokines can be used to distinguish between physiological and pathological hypertrophy42. We show that immune cell presence can also be used in the same manner. Targeting T cell-mediated responses made it possible to interfere with cardiac remodelling. This is in contrast to unsuccessful attempts to limit pathology by targeting cytokines, which have proven to be more elusive targets5,11.
A main clinical feature of pathological cardiac hypertrophy is fibrosis. Fibrosis formation in other contexts requires the combined action of Th2 cells and innate immune cells19,43. In the TAC model we identified an initial M1-polarized innate response, which we speculate subsequently switches to an M2/Th2 polarization. This agrees with studies reporting worse HF in BALB/c compared with C57BL/6 mice, attributed to a Th2-bias of the former strain13,44. We demonstrated the presence of T cells in cardiac biopsies from human HF patients. Moreover, recent evidence shows that genetic deficiency of T cells improves symptoms in the TAC mouse model45,46. These findings, collectively, make a strong case for attempting to regulate T cell-mediated responses in order to combat HF.
Immunosuppressive regulatory T cells (Treg) can block deleterious or unwanted responses25. Intriguingly, evidence has linked Treg deficiency with chronic HF47. We detected the presence of Tregs, via the expression of their genetic marker Foxp3, in TAC mice, but only at 8 weeks post-surgery (Supplementary Fig. 6f). This may be an indication of a natural immunosuppressive attempt that occurs too late to block the pathogenic immune response48. There have been attempts to utilize Treg adoptive cell therapy in models of HF49,50. However, cell therapy is a promising procedure that still needs refinement before it can move to clinical use. Treg can also be activated via super-activating anti-CD28 antibodies, which have been utilized in models of cardiac repair after myocardial infarction51,52. Yet past clinical trials with super-activating anti-CD28 have activated pro-inflammatory memory T cells, with near-lethal consequences for the patients53. Searching for a more readily translatable solution, we utilized abatacept, a fusion protein based on CTLA-4. Treg suppress via surface-bound CTLA-4 as well as soluble IL-10 or TGFβ, inhibiting the function of both innate and adaptive immune cells25. CTLA-4 inhibits T cell function by blocking the ability of T cells to become costimulated. CTLA4-Ig fusion abatacept is easily administered and already in clinical use to suppress autoimmune responses14.
We chose to utilize the TAC mouse model of HF15, which leads to Heart Failure with reduced Ejection Fraction. As no model reflecting the characteristics of Heart Failure with preserved Ejection Fraction has been fully consolidated, TAC remains the most commonly used model for the experimental study of HF54,55. It should be noted that any inflammation induced by TAC surgery per se rather than the constriction may not be fully controlled by the sham operation. Having stated this, as Supplementary Fig. 1b suggests, the surgery-induced inflammation in the sham controls is not negligible.
We demonstrated that abatacept reduced the severity of cardiac pathology and delayed the progression of symptoms of overload-derived cardiac pathology. Our aim was to demonstrate that immunity has a contributing (and targetable) role in the development and maintenance of HF. The presence of T cells in biopsies from patients suffering from either lamin A/C cardiomyopathy (associated with Heart Failure with reduced Ejection Fraction, similarly to the TAC model, yet caused by genetic defects), or aortic stenosis (driven by pressure overload, similarly to the TAC model, yet frequently associated with Heart Failure with preserved Ejection Fraction) offers hope for the theoretical applicability of our approach in the clinic. Translation to the human setting will need further exploration.
Abatacept is known to inhibit T cell activation and proliferation14 by blocking costimulatory ligands CD80 and CD86 on antigen presenting cells (dendritic cells, B cells and macrophages)39,56,57,58. Despite early contrasting data, abatacept has been shown not to act via induction of signals in dendritic cells59,60. Yet, as it interacts with macrophages and B cells, it is not surprising that it can directly inhibit monocyte/macrophage activation and function33,61 and B-cell function34,39,57,58. The functions of macrophages and B cells affected by abatacept are related to T cell-dependent responses33,34,39, possibly as these functions involve CD80/CD86.
In agreement to the known mechanisms above, we found that abatacept inhibited T cell responses in vivo (Fig. 4a,b), including in heart-draining lymph nodes, where T cell activation appears to be initiated (Fig. 2e). We also observed an inhibition of cardiac macrophage activation and maturation (Fig. 4c–e). Further, we identified the induction of anti-inflammatory cytokine IL-10 (Fig. 4f), which was necessary for the protective effects to occur and which could be produced by B cells after in vitro treatment with the drug (Supplementary Fig. 6c). Il10-sufficient B cells appeared to be sufficient to rescue the loss of cardioprotective effects in Il10 KO TAC-operated animals treated with abatacept (Fig. 5g,h). The schematic outline of this combined inhibition of pro-inflammatory T cell/macrophage functions and induction of anti-inflammatory signals in B cells is given in Fig. 6. As shown, T cell45,46 and monocyte/macrophage35 pro-inflammatory function is cardiotoxic. Upon abatacept administration, the mechanisms described above may be acting in parallel. Several caveats must be mentioned: the finding that immune-mediated events can drastically change the outcome of disease does not render inflammation the only aspect that can regulate HF pathology. Second, it should be noted that IL-6 is produced by stressed cardiomyocytes, initiating the inflammatory response that accompanies HF3,6,7,9. Our data suggest that inhibition of T cell and macrophage function, which lie downstream of the initial inflammation, triggers compensating anti-inflammatory IL-10 expression but may not be significantly affecting the IL-6 production by cardiomyocytes. Finally, abatacept has been shown to induce regulatory T cells62, yet we did not observe any significant induction of Foxp3 mRNA expression in our system (Supplementary Fig. 6g).
The benefit conferred by abatacept treatment may be that it targets T cell costimulation and thus their optimal activation. T cell activation could be relevant for the chronicity12 of the underlying cardiac disease. As a drug already in clinical use, abatacept may be more translationally relevant than other means of targeting T cells currently being explored for the treatment of HF. Further, targeting costimulation requires the targeting of CD80/CD86-bearing macrophages and B cells, which contributes to the therapeutic effect, affecting T cell-associated B cell and macrophage responses.
IL-10 is directly cardioprotective and antifibrotic19,41. IL-10 was necessary for the cardioprotective effects of abatacept, and for the suppression of T cell expansion (Fig. 5a). Yet IL-10 acts downstream of the administration of abatacept. Thus, the regulation of IL-10 induction will be dependent on localization and abundance of the targets of the drug. Abatacept, even when B cells and macrophages are its direct targets, is known to affect only T cell-associated responses33,34,39. Abatacept did affect T cell activation systemically (as shown in Fig. 4a) but, extrapolating from the data in autoimmune pathologies cited above, it may not affect T cell-independent innate immune responses, even if its action is dependent on IL-10. IL-10 is a very potent anti-inflammatory cytokine; clinical trials for its use have yet to succeed63. Its direct administration could possibly block T-independent responses resulting in more severe immunosuppression. Thus, we speculate that abatacept, given its proven clinical safety profile, may be more translationally relevant compared with IL-10 administration, as a potential HF therapy tool.
Taken together, our findings demonstrate how an FDA-approved drug inhibiting pro-inflammatory T cell function, along with effects on macrophages and B cells, yields significant therapeutic benefits in a model of HF. This occurs as an adaptive immune response may be causatively linked to the pathogenesis of pressure overload-induced HF. An immune response driven by cardiac pressure overload could be an unwanted consequence of an immune system evolved to deal with pathogen infections. It may be that the body cannot distinguish between infection- and pressure overload-induced stress signals, and hence initiates a deleterious response. Fortuitously, the link between immunity and HF also creates an opportunity: validated therapies for treating immune-mediated ailments exist and are already in clinical use. They could be repurposed as potential tools in the fight against HF, paralleling the rationale of recent promising studies in other pathologies64.
All procedures were performed in compliance with national and EU legislation, and Humanitas Clinical and Research Center and Norwegian University of Science and Technology regulations.
Procedures were performed according to ref. 15. In detail, TAC was performed on 8–10-week-old male C57BL/6J mice (Charles River, France) and on 8–10-week-old male C57BL6/J Il10 KO mice (Jackson Laboratories, US). All animals were screened before operation via echocardiography to establish their baseline. Mice were anaesthetized by intraperitoneal injection of a mixture ketamine (100mgkg−1) and xilazine (10mgkg−1). The chest cavity was opened by a small incision at the level of the first intercostal space. After isolation of the aortic arch, a 8–0 Prolene suture was placed around the aorta and a 27G needle was laced in between. The needle was immediately removed to produce an aorta with a stenotic lumen. The chest cavity was then closed with one 6–0 nylon suture and all layers of muscle and skin closed with 6–0 continuous absorbable and nylon sutures, respectively. A sham group, undergoing surgery without aortic banding, was used as control.
A Vevo 2100 high-resolution in vivo imaging system (VisualSonics Fujifilm) with a MS550S probe 'high frame' scanhead was used for echocardiographic analysis. Mice were anesthetized with 1.0% isoflurane for M-mode imaging. Pressure gradients (60 to 90mmHg), an index of biomechanical stress, were determined by echo Doppler on all animals that underwent TAC surgery.
Starting 2 days or 2 weeks after TAC/sham surgery, mice were intraperitonally injected with either 100μl PBS, 200μg Human IgG Isotype Control (Novus) or 200μg CTLA-4 Ig (Abatacept) in 100μl of PBS, three times a week, for up to 4 weeks. Abatacept is a human CTLA-4-Ig fusion, though due to the high (75%) similarity between human and mouse CTLA-4, it also functions in mouse28,29,65,66,67. These studies demonstrated in vivo efficacy (in different pathological contexts) using a dose range 100-400μg per mouse, in most cases administered every 2 days. These studies, collectively, identify a range of abatacept dosing that is functional in mouse. As 200μg per mouse every 2 days was both the median dose of the published mouse studies, as well as (at about 8mgkg−1) very similar to the human dose used in Rheumatoid Arthritis patients (8–10mgkg−1), we selected this dose as the most 'translationally relevant'.
Wild-type B and T cells were isolated from 10–12-week-old male C57BL6/J male mice, respectively, with B Cell Isolation Kit and Pan T Cell Isolation Kit II (Miltenyi Biotec) on an AutoMACS. Purity was assessed by staining with anti-mouse CD3 (145-2C11, BioLegend) or anti-mouse CD19 (eBio1D3, eBioscience), and analysed by flow cytometry. C57BL6/J Il10 KO male mice, before basal echocardiography screening, were injected intravenously with 2 × 106 WT T or B cells. Mice underwent TAC surgery and were injected with abatacept starting on day 2 after surgery.
The severe cardiomyopathy patient samples (HF LVAD) were obtained from patients suffering from lamin A/C mutations, causing dilated cardiomyopathy and HF (HF LVAD 1M). A subset of these carried a second mutation in titin (HF LVAD 2M), leading to a more severe dilated cardiomyopathy. All samples were obtained after informed consent according to the study protocols approved by the ethics committee of the University Hospital of Verona23. Aortic stenosis ventricular samples were also obtained after informed consent according to the study protocols approved by Humanitas Research Hospital ethics committee.
Left ventricles were snap frozen in liquid nitrogen after collection and stored at −80°C. Tissues were homogenized in 1ml of PureZol RNA isolation reagent (Biorad) with GentleMACS and GentleMACS M Tubes (Miltenyi Biotec). After isolation of the aqueous phase with chloroform, RNA was extracted using RNeasy Mini Kit (Qiagen). The same amount of RNA was retrotranscribed with the High Capacity cDNA Reverse Transcription kit (Applied Biosystems). Real-time qPCR reactions were performed using TaqMan Probes and TaqMan Universal Master Mix on a REALTIME AB 7900HT cycler (all Applied Biosystems). The following TaqMan gene expression assays were used: Rn18S (Mm03928990_g1) as internal control, Cd3e (Mm005996484_g1), Foxp3 (Mm00475162_g1), Itgam (Mm00434455_m1), Tnfα (Mm00443260_g1), Il4 (Mm00445259_m1), Il17 (Mm00439618_m1), Ifng (Mm01168134_m1), Tgfb1 (Mm01227699_m1), Il10 (Mm00439614_m1), Il6 (Mm00446190_m1), Il1b (Mm00434228_m1), Ccl2 (Mm00441242_m1), Ccl4 (Mm00443111_m1), Ccl5 (Mm01302427_m1), Cxcl10 (Mm00445235_m1), Cxcl11 (Mm00444662_m1). Expression of genes encoding for Brain Natriuretic Peptide (Nppb), Atrial Natriuretic Factor (Nppa) and Myosin heavy chain β (Myh7) expression was tested with primers (IDT) using Sybr Select Master Mix (Applied Biosystems) on a ViiA7 (Applied Biosystems) instrument. The sequences are listed in Supplementary Table 3.
Male Akt Tg mice22, which constitutively overexpress the active E40K Akt mutant (Akt-E40K) were used at 8 weeks of age.
BKS.Cg-m +/+ Lepdb/+db mice67 are heterozygous for the leptin receptor mutation but display a wild-type metabolic phenotype when fed on a normal diet. We utilized 8-week-old male mice that were arbitrarily assigned to one of two groups: sedentary and exercise trained 70min per day, 5 days per week, for 8 weeks. The training was performed as running on an inclined (25°) treadmill, starting with 10min warm-up at ~50% running speed before 60min interval training alternating between 4min at 85–90% of maximal oxygen uptake and 2min at ~50% running speed. Training speed was adjusted at least weekly in order to keep the same relative training intensity. Before and after the intervention period, the mice performed an individualized ramp (90–120s on each step) treadmill protocol on an inclined (25°) treadmill in a metabolic chamber to determine maximal oxygen uptake. Due to the difference in genetic background (BKS), all analyses of these mice were performed comparing them to their matching controls, so as to avoid genetic background-specific effects.
Mouse heart samples were fixed in 4% formalin at 4°C, paraffin-embedded and sectioned at 4μm. The slides were stained with Azan's trichrome for collagen (BioOptica). Slide images were digitalized and five fields for mouse sections and ten fields for human biopsies analysed to quantify fibrosis, with an image analysis program (ImageJ). Cardiac fibrosis was assessed by measuring the Azan's trichrome-stained area as a percentage of total myocardial area. For immunohistochemistry analysis sample sections on slides were deparaffinized and hydrated through a descending scale of alcohols. Antigen retrieval was performed using DIVA (Biocare Medical) for mouse samples and W-Cap (Biocare Medical) or EDTA 0,5M pH8 (Sigma Aldrich) for human samples. Sections were cooled and then washed with PBS (Lonza) containing 0.05% Tween 20 (Sigma). Endogenous peroxidase was blocked by incubation with Peroxidase I (Biocare Medical) for 20min at room temperature (RT) and nonspecific sites were blocked with Rodent Block and Background Sniper (Biocare Medical) for mouse and human samples respectively 20min at RT. The sections were then incubated for 1h at RT with rat anti-human CD3 (Serotec) diluted 1:1,000 or AIF-1 (Wako) diluted 1:250 or polyclonal rabbit anti-human CD3 (Dako) diluted 1:50, washed, and incubated for 30min at RT with rat-on-mouse HRP polymer (Biocare Medical) or with Mach1 HRP polymer (Biocare Medical) or with Envision+System anti-rabbit HRP (Dako). Finally, sections were incubated with DAB (Biocare Medical), counterstained with haematoxylin, dehydrated through an ascending scale of alcohols and xylene, and mounted with coverslips using Eukitt (Fluka). All samples were observed and photographed with a microscope Olympus BX53 with a digital camera.
Sample sections on slides were deparaffinized and hydrated through a descending scale of alcohols and TUNEL assay was performed (Click-it plus TUNEL assay C10617, Life technology).
Total splenocytes were purified from spleens of 8-week-old C57BL/6J mice. T cells were depleted using magnetic beads on an AutoMACS (Miltenyi Biotec). Total splenocytes or T cell-depleted splenocytes were stimulated with 2μgml−1 of anti-CD3 and/or 5μgml−1 LPS (Sigma Aldrich) and cultured with 20μgml−1 abatacept, IgG isotype control or nothing. After 48 or 72h of culture, Brefeldin A (eBioscience) was added during the last 4h of culture and splenocytes were prepared for fluorescence-activated cell sorting analysis.
Hearts were collected from 1–2-day-old CD1 pups and digested with collagenase. Cardiomyocytes were then separated from fibroblasts by preplating twice for 1h and through centrifugation. Cardiomyocytes were than plated over gelatin, serum-starved and treated with 100μM phenylephrine. Four hours after the addition of phenylephrine, 20μgml−1 of abatacept were added to the culture for 44h. Cardiomyocytes were harvested in PureZOL (Biorad) for RNA extraction and gene expression analysis.
Single cell suspensions from spleens and lymph nodes were obtained via passing through 70μm cell strainers in cold PBS−/−. Hearts were collected and digested with Liberase TM (Roche). Erythrocytes were removed with lysis buffer (BD Biosciences) from spleen and heart cell suspensions. Cells were stained at the following dilutions of stock reagents: Live/dead Aqua Fluorescent Reactive Dye 1:1,000 (Life Techonologies), anti-mouse CD16/32 1:100 (2.4G2, BD Pharmigen), anti-mouse 1:100 CD45 (30-F11, eBioscience), anti-mouse 1:100 CD3 (145-2C11, BioLegend), anti-mouse 1:100 CD19 (eBio1D3, eBioscience), anti-mouse 1:100 CD11b (M1/70, Biolegend), 1:100 CD11c (Bu15, eBioscience), F4/80 1:100 (CI:A3-1, Serotec) in Supplementary Fig. 5d, F4/80 1:100 (BM8, eBioscience) in Fig. 5d,e and Supplementary Fig. 5c, IL-10 1:80 (JES5-16E3, eBioscience), FoxP3 1:100 (FJK-165, eBioscience), Ly6C 1:100 (HK1.4, eBioscience) or anti-CD25 1:100 (PC61.5, eBioscience). An eBioscience intracellular staining kit was used were applicable. Samples were acquired on a fluorescence-activated cell sorting Canto II (BD) and analysed with FlowJo10.
Statistical analysis was performed in GraphPad Prism. All data sets were tested for normal distribution with normality tests before proceeding with parametric or non-parametric analysis. Grubb's test was performed in order to exclude spurious outliers. Statistical significance was tested using unpaired t-test, one-way analysis of variance (ANOVA) with Tukey post-test and two-way ANOVA with Bonferroni post-test for data sets with normal distributions. Statistical significance was tested with Mann–Whitney test and one-way ANOVA with Dunn's post-test for data sets without a normal distribution. Fisher's exact tests were used in the analysis of collagen deposition, testing for the presence or absence of collagen stain.
All the relevant data are available within the manuscript and from the authors upon request.
How to cite this article: Kallikourdis, M. et al. T cell costimulation blockade blunts pressure overload-induced heart failure. Nat. Commun. 8, 14680 doi: 10.1038/ncomms14680 (2017).
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Supplementary Figures and Supplementary Tables.
The work was supported by the E.R.C. (CardioEpigen; 294609), Fondazione Cariplo (2015-0573) (to G.C.), The Fondazione Veronesi (to M.K.), Fondazione Cariplo (2014-1184) (to M.K. and D.C.) and the Italian Ministry of Health (GR-2013-02355011) (to M.K.), the Norwegian Council on Cardiovascular Disease, the K.G. Jebsen Foundation and UNIKARD (to T.O.S.). We wish to thank B. Cassani, A. Villa and R. Bonecchi/M. Locati for their generous gift of reagents; A.G. Betz, K.G. Andersen, M. Latronico, D. Morone for useful advice, M. Vacchiano for technical assistance. Abatacept was a gift of Bristol Myers Squibb (USA).
The authors declare no competing financial interests.
Author contributions M.K. designed experiments, interpreted results and wrote the paper, E.M. performed ex vivo experiments and prepared figures, P.C. performed in vivo experiments, C.S., G.R., C.M.G., D.V., F.R., A.M.O.B., T.O.S., A.F., G.F., E.D.P. contributed to experiments, L.E., C.R., D.C., R.P. contributed to design of experiments and interpretation of results, G.C. designed experiments, interpreted results and wrote the paper.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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2 bedroom house for saleElgin Avenue, Maida Vale, London, W9
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Greene and Co are delighted to present this bright, split level apartment set on the first floor of this converted Victorian building. Offering two double bedrooms the property also benefits from high ceilings and large sash windows is is being sold on a chain free basis. Elgin Avenue is an attractive residential street within easy reach of a wide selection of shops, cafes and restaurants in Maida Vale whilst the green open spaces of Paddington Recreational Ground are moments away. The property is also a short walk from both Maida Vale and Warwick Avenue Tubes (Bakerloo line) as well as Westbourne Park (Hammersmith and City and Circle Line).
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When: Sun., March 27, 6 p.m. 2016
The internationally acclaimed Jazz Ambassadors of Washington, DC will continue its long tradition of presenting free public performances when it appears at The Tower Theatre.
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Mindy has been in the field of education for more than 20 years, teaching, writing curriculum design, and serving as a principal. She loves working with kids and they love working with her. She came up with the idea of an educational cooking camp when she observed how much children in the Temple Israel Religious School enjoyed cooking. When she offered up explanations about a food's origins and the customs behind preparing the food, she watched the children's engagement grow.
"Cooking and eating takes on a different meaning when you understand the back story," said Mindy. "Learning a little bit about a country, then preparing that country's food truly enriches the cooking lesson, and makes the eating experience that much more delicious!"
A California native, Victoria has avidly explored the cultivation and preparation of artful, nourishing food since childhood. While studying Agroecology at UC Santa Cruz, she engaged her student community with seasonal eating as an Events Coordinator for the university-affiliated Program in Community and Agroecology. She led informative gardening seminars, culinary workshops, and weekly meals featuring home-grown fare that reflected the diversity of the community. While budgeting activities, she managed the procurement and use of communal goods, created quarterly production schedules, and organized many successful fundraising events. To ensure the continuation of the program, she composed several rewarding grant applications. Her dedication to sustainable food led her to promote local, whole-animal eating with Marin Sun Farms where she managed the farmer's market division. She successfully built a strong sales team, composed engaging educational and promotional materials, supported online commerce and presence, and maintained harmonious professional relationships. The diversity of the produce she beheld at market has inspired her to continue celebrating the cuisines of myriad cultures.
Madeleine was born and raised in the Bay Area and has always loved learning, working with children, culture and food. For the past three years she has worked in human relations at Whole Foods Market. This work exposed her to opportunities for teaching and travel, and allowed her to become involved in company programs abroad where young women in desert areas of India received educational programming. Madeleine's passion for education is also reflected in her future school plans for getting her master's degree in educational systems.
Born and raised in Oakland, Kate has been exposed to so many different cultures which lead her to pursue a degree in History. She loves to watch children learn and grow. Her first job was an internship at the Lawrence Hall of Science at the age of 14 where she could help young children learn the joys of sience. She is fascinated with all the cultures the world has to offer, especially all the different kinds of food. Her love of different cultures took her to Korea where she taught English to grades 3 - 12.
Ariel is a mom with a variety of talents and interests. She has always been fascinated by different cultures and their different cuisines. As a young girl, she was introduced to many different types of food, tasting and cooking Ethiopian, Hungarian, Japanese, Israeli, and Italian dishes.
A Philadelphia native, Kat grew up surrounded by different cultures and cuisines, including her family's Sicilian seven fish dinner at Christmas time. Coming from a family of cooks, and a twin brother who is a professional chef, it's no wonder she has always found passion and solace in cooking and baking. Eventually, she moved out to the Bay Area to bake at Acme Bread, ending up in Oakland. For the past 8 years, she has been a professional nanny, reconnecting with her love of children and childcare. Kat has a biology background and is currently pursuing her BA/BS in Education and Nursing in the evenings. Kat's love and excitement for exploration and experimentation with food and cuisine is optimistic and endless. She is shameless about getting goofy and involved with every student, while her energetic and jolly personality is sure to engage every meal and challenge.
Natalie grew up in the Bay Area and graduated from SJSU with a Bachelor's Degree in Business. She teaches cooking with the children at the Jewish Community Center in Oakland. In her free time she enjoys spending time with her husband and her two dogs, Butter and Biscuit. Other than her passion for cooking and baking, Natalie spends her free time exploring different foods, traveling, kick boxing, and working on her cookbook.
Meredith's journey with expanding her palette began in her mid-twenties. I was 22 years old the first time I tried an avocado! I truly believe it's never too late to try new things and I have seen how food has a way of opening doors and expanding other aspects of enjoying life. I love trying new foods and flavor combinations that I never thought would work together. I first started traveling abroad to Europe and then to South East Asia where I visited some amazing countries and made wonderful friendships. Most recently, I have been exploring the United States and learning about the unique lands and cultures that surround me.
Talia has been part of the Cooking Round the World program since its inception. As the daughter of the director, she influenced the shaping and formation of the program. She is "a natural" with children as a teacher and a mentor and is studying education at Cal Poly to official become a credentialed teacher. Along with her mom, she has visited 43 countries around the world, taking cooking classes wherever she goes, visiting food markets, and gaining firsthand knowledge about cultural likenesses and differences. She is delighted to share what she knows and inspire enthusiasm with the Cooking Round the World campers!
Samantha describes herself as a food geek. She loves to learn about every aspect of food – from its origins and traditions to its farming and production, and most importantly, its consumption! Her professional food adventure began modestly in a dive bar, frying chicken and BBQ-ing ribs. After earning a culinary degree, her inquisitive nature took her to a corporate test kitchen where she created her categories of salad dressing, among other pursuits. Most recently, her work at a high-end restaurant allowed her to use local ingredients to create a variety of dishes that were responsibly sourced, beautifully rendered and delicious. Samantha has followed her insatiable curiosity into every corner of the food industry and now she is ready to share that knowledge with her students.
Sawittri grew up in Malaysia, where friendships and family ties are strengthened by a common love for good food. Amongst her family and friends, it is very common for plans to be made about where and what to eat for lunch while breakfast is being enjoyed! In fact, the first thing Malaysians say when they meet is not "How are you?" but "Have you eaten?". The answer is irrelevant - you go have a meal anyway! As a spouse to an internationally mobile employee, Sawittri has lived in Canada, Southern California and now the East Bay. She has also travelled to different parts of Europe, South America and Asia and has been fortunate to sample many beautiful local dishes. Sawitttri holds a degree in Education and a Masters in English as a Second Language (ESL) and has served as an ESL teacher, a teacher coach and most recently an ESL tutor.
Sara Ketcham is a Bay Area native, and she is committed to a life of community building and education. She currently teaches World History and English at Oakland Technical High School. For her undergrad, she majored in Global Studies at UCLA, a degree that looks at international development and globalization. Sara believes there is great value in learning about the cultures and experiences of people around the world! Her love for cooking and learning recipes from other cultures began during a semester in Italy, where every meal is a delicious work of art. She has continued to learn recipes from French, Swiss, and Indian housemates and is excited to join the staff of Cooking Round the World!
Caroline has enjoyed cooking diverse foods for many years and has worked with elementary school-aged for 4 years. She is thrilled to merge her two passions or teaching and cooking. She believes that cooking foods from different parts of the world is an invaluable experience for children and adults alike, Leaning about different cultures, making connections, and participating in a hands on activity like cooking fosters openness and acceptance.
David's passion for cooking and food stems from his childhood experiences in the kitchen and around the dinner table. Meals became not only a source of nourishment but shared expressions of creativity and knowledge. This sparked a sense of wonderment that made him love learning in and outside the classroom. He hopes to share with children that cooking can be a positive outlet for personal growth that is made to be shared with others.
Jamie May is a fourth-year graduate student studying clinical psychology. Within this program, he has received training in psychodynamic therapy, social justice, and community mental health. He previously worked as a chef, a musician, and a camp counselor. He has also had several opportunities to teach as a tutor and teacher's assistant. He is particularly enthusiastic about the opportunity to help students learn about various cultures and cooking techniques, drawing upon his own personal experiences and the weekly lessons provided by Cooking Round the World.
Jowelle is an upbeat, passionate and easy going chef who can make herself feel at home in any kitchen. As a recent graduate from the Art institute of San Francisco majoring in baking and pastries she has learned how to integrate her passions into a career. She hopes to set the pathway for other young kids to foster their own unique gifts beginning with taking simple ingredients and creating masterpieces. Jowelle's passion for integrating culinary practice into the lives of young children is driven from her own personal struggles with a learning disability that challenged her in every classroom except for the kitchen. Her experience with baking as a child gave her all the right ingredients to create mouthfuls of memories for friends, loved ones and now children.
Vidya is an extremely versitile chef with positive energy and a great working knowledge of cuisines from around the globe. She studied at Le Cordon Bleu, worked in fine dining for many years, and taught cooking classes for both adults and children alike. Most of all, she loves cooking, teaching, and sharing her knowledge and passion with her students in a fun, yet informative way.
Jonna is a Martinez ca native born into a family of butchers and caterers. She is a former equestrienne and athlete but even after all the glitz and glam of horse showing, tournaments and exhibitions, her heart remained true to the family's roots! She has a great passion for food, children and instruction!
Graduating from California Culinary Academy in 2001 Chef Leila went on to be a private chef specializing in custom menus for Doctor recommended diets co-creating with a licensed nutritionist. Chef Leila developed the gourmet panini menu at The Epicurean Trader, a market with a mission to bring great handcrafted artisan products at small batch local level.
Born and raised in the East Bay, Clarity Countryman was encouraged to explore worldly cultures from a young age. Her mother's enthusiasm for world music had a profound influence on her childhood, as she grew to love the rhythmical sounds of West Africa. She spent a majority of her upbringing in the kitchen with her father, which is where her passion for cuisine was sparked. Since earning her degree in Marketing, Clarity has managed a coffee shop, cared for neighborhood children and served in a fine dining establishment in San Diego before returning to the Bay Area. She did this move by way of a culinary adventure through Italy, Greece and Spain in the summer of 2014. Some of the best memories from this trip were sharing a dinner with Senegalese cave-dwellers in the hills of Granada, Spain and visiting the birthplace of balsamic vinegar- Modena, Italy. In her spare time, Clarity enjoys sharing new recipes with her 3 younger siblings and camping with close friends and family.
Colin's first memory of cooking was from when he was 4 years old, "helping" his father make chocolate chip cookies by stealing chocolate chips every time his dad turned around. For the past 9 years, he's been cooking professionally in a variety of settings, from sandwich shops, to fine-dining restaurants, to modernist kitchens. He loves making food that's an expression of creativity and technique, but also warmth and hospitality. He's had many opportunities to experience first-hand how food and cooking can bring together people who don't initially seem to have much in common in really wonderful ways. He'll also still probably steal your chocolate chips if you're not looking.
Serena has been a camper at Cooking Round the World 3 times and has fallen in love with the program. She was inspired by all the recipes she learned while at camp. Each day she would learn something new and she would always have so much fun making it on her own at home. Serena has always loved cooking dishes with her dad because it gave her a sense of independence. She loves baking in general, but when her cousins come over, that's all she wants to do with them. Going to new places and traveling the world is something Serena loves because she gets to see the different cultures and cuisines that people other than herself have. To her, cooking is a way to put random ingredients together and turn them into your own unique creation.
Fredrikka loves food and loves to try different foods because when she does she begins to wonder about the history of the dish. For every new dish, she tries to learn about its culture. This is important because with that knowledge she is able to teach others things about cultures.
Food has been a part of Nicky's life for as long as she could remember. She's seen how food can effect people both physically and emotionally and how it ties into our history as humans. She believes if there's one sure-fire way to connect with people from all walks of life: it's through food! She really wants to pass the knowledge she's gathered in her time to others so they can also experience the magic of food.
Regan was a camper at Cooking Round the World summer camp since she was 10 years old! She will be starting high school in Oakland this fall, and decided to come back to Cooking Round the World this summer as a Counselor in Training. Regan loves soccer, working with kids, and of course, cooking! She loves to bake at home, and will often make meals for her family. Some of her favorite recipes from the program are Hungarian dumplings and Forgotcz Fank -- Hungarian twisted doughnuts. She hopes to gain further experience in the kitchen, and to open doors to new cultures and experiences!
At a very young age, Sofia set a goal for herself to be able to do everything in the kitchen by herself. She started cooking with her mom, dad and sister, and eventually began making desserts for family birthdays and friends. At 10 years old, she attended Cooking Round the World summer camp. Now at 14 she decided to return, now as a Counselor in Training. She is open minded, creative, energetic, and much more! Cooking is is one of her greatest passions, and she is excited to share her passion with others!
Food is important to keep us alive, but cooking and trying new foods is a fun way to make it so much more than that. Chiara loves to cook because it feels very rewarding to turn simple ingredients into something special and delicious. She also loves cooking and eating foods from new places. Food is one great way to learn about another culture and lifestyle, and trying new things opens everyone's minds to new ideas.
Lauren was born in Berkeley and has lived both in the Bay Area and San Francisco. She's loved cooking and helping out in the kitchen since she was very young and is always looking to cook new and culturally diverse dishes. Being multi-ethnic, she has had the opportunity to cook and experience dishes ranging from soul food to Spanish and Croatian fares. Lauren plans on deferring her first year of college to study abroad where she plans on capitalizing on her interest in culturally diverse cooking and food fusion. In her free time she enjoys spending time with her two-year old nephew, shopping and hanging with friends.
Taylor has always loved cooking and trying out new recipes at home. Although her kitchen is very tiny, it still is her favorite place in the house because she can be creative and try new things. She enjoys being with kids because they tend to be really funny and have a really big imagination. When it comes to traveling, she has been to Los Angeles, New York, and Mexico. Even though she goes there often, it's still really enjoyable because she gets to try all the different foods! Last summer Taylor was a CRTW camper and she is delighted to return this year as a CIT.
Krystal loves food. It's as simple as that. She loves food just as much as she loves people. She began her culinary journey during high school when her mom encouraged her take a culinary class that initially she had no interest in taking. After the first few weeks in the class she absolutely fell in love with the art of cooking and everything associated with it. Thereafter she completed two years in culinary academy and progressed to become a pastry chef and line cook in various locales around North County. She has a passion for food like no other, and is SO excited to share the culinary journey that she was able to experience with kids, in hopes that they are able to find the same joy in the kitchen as she has.
Jess was born in a small country town in Australia. From a young age, she has loved food. For Jess, cooking is about family and community. She is a developmental psychologist and completed her Ph.D. in 2014. Her research explored social understandings in children cross-culturally. During her years of study, Jess traveled the world and worked with children and families in many countries. She lived in the Netherlands, traveled throughout Europe and spent time in Iran and China and now she is living in San Diego. Travel has allowed Jess to experience different cultures and try new food and local delicacies. Jess believes food has the power to bring people together to celebrate culture and diversity and is thrilled to be a part of Cooking Round the World!
Tanya is passionate about delicious food, creativity, teaching, and building life skills. She teaches 5th grade at Heritage Flex Academy, and recently completed her Masters and Teaching Credential from Alliant International University. She brings her experience in hospitality, teaching at a variety of summer camps, and lots of excitement to the team! While she grew up in San Diego, she has called San Francisco her home for the past 5 years, and loved exploring the tastes and adventures of the city-especially, at the Ferry Building. Being back in San Diego, she has been growing her passion for gardening, especially watching her herbs and strawberries grow, and dreaming up new recipes for seasonal produce. She is excited to whip up lots of tasty treats with Cooking Round the World!
Working with food is Jessica's passion. At a young age she knew she wanted to be a chef, but didn't know how it would impact her life in such a wonderful way. After working for major hotels and corporate kitchens, she uprooted her life and moved to the mid-west to work with children and teach them about using fresh ingredients. She planted herb gardens, vegetable beds, and after a few months she started putting the ingredients into lunches and even taking some of the harvests home! She believes teaching through interaction and hope that those interactions lead to powerful experiences. She is "spiced up" to be apart of the Cooking Round the World team!
Jenny is originally from the Washington D.C.'s suburbs but moved to San Diego for the sun and warm weather. She grew up with a passion for food thanks to her Haitian roots. She attended an international French school in D.C. During undergrad, she moved to Rome, Italy, and her passion for cooking and food only grew. She now travels to experience new cuisines and to experience new cultures. She is currently completing her Masters Degree in Nutrition Education at American University. In her spare time, she teaches nutrition education workshops, volunteers at local food distributions, hikes and relaxes by the beach.


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Life skills
LIFE SKILLS Sequencing Mats
Life Skills Room
Hygiene Life Skills Unit (Special Education & Autism Resource)
10 pack practice worksheets - Will need to use this in class next year!
Empowered By THEM: Getting Organized - Curriculum By Subject
Putting Away Groceries - Life Skills, Interactive Book. Here is a fun life skills lesson on putting away groceries. There are 33 food pieces and three options of were you can put them, 12 pantry, 9 freezer, and 12 refrigerator. Get the free download at: https://www.teacherspayteachers.com/Product/Putting-Away-Groceries-Activity-Book-1436613
100 Basic Life Skills •
What Do I Need? {meal prep, life skills, independence}
14 Life Skills Worksheets
Functional Life Skills: Laundry Lessons & Apps. Download, print, and go! Speech Room News
Life Skills Centers: Year Long Life Skills Bundle
Teaching Children How to Fill Out Forms
Life Skills - Kitchen Safety
New Work Task Boxes for Special Ed
FREE Printable Life Skills Checklist for Kids
Life Skills Problem Solving: In the Community


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4 Comments
Alvin and the Chipmunks: The Squeakquel
2nd to 8th Apr
Pop sensations Alvin, Simon and Theodore are back for their second big screen outing.
Astro Boy
9th to 15th Apr
Set in a futuristic Metro City and stars Astro Boy, a young robot with incredible powers.
16th & 17th Apr
Magical animated fantasy adventure based on the cherished Hans Christian Andersen tale of 'The Little Mermaid'
24th & 25th Apr
Animated comedy adventure from Pixar.
Films can vary - its Alvin and the Chipmunks at York.......and although its good value, I wouldn't say it was hot as its always this price, every weekend and school hols.
shame this isn't on at westwood cross, thanet but you reminded me about checking to see what's on so thanks :thumbsup:
thanks for the heads-up, have changed op.
Thanks - politeness is so rare on here - I've added some heat and rep :thumbsup:


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My husband and I are in Vevey for his work. When his co-workers found out that it was his birthday they wanted to take him out, so last minute we found ourselves with a party of 10. We wanted fondue and had heard good things...More
71 - 80 of 90 reviews
My husband and I are in Vevey for his work. When his co-workers found out that it was his birthday they wanted to take him out, so last minute we found ourselves with a party of 10. We wanted fondue and had heard good things...More
Great fondue, friendly staff and the way the fondue is delivered is absolutely unique. You have to be there... Let's just say that some tongue in cheek military setup is involved.
the food is average, but there is very laud military music and a person turning around with a swiss flag each time they serve fondue! It can interrupt your meal 10 times or more, absolutely not a pleasure.
Fondue fondue fondue....and some folklore...good value for fun....good simple local wines (beware your ulcers...)...
Incredible - the manager/head waiter was completely drunk or intoxicated with something. He was extremely arrogant & rude from the start and was very sarcastic and bitter from the moment we entered he said he didn't like my shoes! I ignored this and thought I...More
We were recommended to this restaurant by our hotel reception who organised the table booking for us. It was a Friday night, so the restaurant was busy, with a lively atmosphere. The fanfare servings of the fondue wore a bit thin after a while, but...More
Quite an interesting atmosphere in the heart of Vevey. I have always had their fondue and never tried the other plates. Fondue is very good, Gruyère and Vacheron cheeses, nice wine and the side dishes are interesting. However I think you shoud stay just with...More
I just came back from eating dinner at Les 3 Sifflets (on the recommendation of the reception at my hotel). It has a fun and quirky atmosphere which I did enjoy. I had the filets de perches, which admittedly was overcooked. I indulged in a...More
Very Swiss. A little over the top with the showmanship in the sense that if you order a traditional Swiss dish like the fondue, it is brought out with full fanfare- servers with trumpet and Swiss flag. Food was not bad either.
It was a total coincidence that we've ended having a dinner at Les 3 Sifflets. The place was fully packed, fondue was excellent but even better was the atmosphere and serving show.
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Sorry for any inconveniences, but I have transferred this rp to @WarriorCatManiacs! Check my rp out there!
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Top Industrial Direct Tech for Home Automation
Automation is something which is currently big business in the technological world. From the earliest beginnings of chair lifts and drawers which opened and closed by themselves, we now live in a world which includes smart homes where everything is automated which can possibly be automated, from motion system doors to the bubble bath. While for many people, this level of automation is enough, technology is rapidly bringing a new level of smart home to our fingertips.
The internet of things, for example, is set to bring interaction with our appliances to new levels. Instead of simply being able to interact without devices via a remote control or mobile device, the internet of things will allow the appliances to speak back to us, letting us know what is happening and how to fix it.
So, what are the current trends in industrial tech application for home automation that we need to be aware of?
Progressive actuators can be used in a variety of different direct automation ideas in the home, from small projects to bigger ones. People mostly know about actuators through their uses in TV lifts and window openers and the like – simple operations which can be performed by simple actuators, giving everybody a much more comfortable home in the process by creating a TV table which can be set to an appropriate height by anybody who happens to be using it at the time.
Actuators can also be used, and are increasingly used, in security systems and the like; they can be attached to cameras to make them easier to use, by improving the range of ground they can cover. An additional draw for actuators in security systems is that the increased movement capacity means that cameras can be put in less obvious to see places, and still maintain their efficacy.
Motion systems
Motion systems are likewise becoming more popular in home automation, and the trend appears to be spreading from one system to the next.
Motion sensors are most popular in lighting systems, as they can save electricity for the homeowner by only having lights on when they sense people in the room who might need light. Naturally this has a lot of applications, not the least of which are security applications; by this we do not simply mean the usual applications of having motion sensor lights keyed to go on when there is abnormal movement in the garden or driveway, but more broadly in terms of having lights coming on in porches and dark doorways when there is someone there. Not only will this make people feel safer, but it will also make it easier for people inside the house to know when there is someone either at the door, or unlocking it.
Motion sensors can also be used for lights which are in less-travelled areas. Rather than have lights which may or may not come on when wanted, have them do so automatically.
Mobile Apps
As a mean to automate your home, mobile apps are perhaps one of the most interesting. There are so many different varieties of app around, particularly since nearly everybody now has access to some form of mobile device, that any homeowner is sure to find something which suits them best.
Home automation apps can link to the various systems in a house, whatever they are, allowing a homeowner to control them from the mobile device of his choice, allowing him the freedom of keeping his hands free for other tasks.
Now over to you
Those are just a few of many advances in home automation. What's your favorite? Please share your thoughts with us.
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Trading Mothers for Daughters
Maternal love goes to the XXX-treme, when lustful mothers lead by perverted example, showing their horny, jealous daughters the joy of unbridled sex. The hungry, gorgeous MILFs, Diamond Foxxx, Dyanna Lauren and Lisa Ann, get their poundings deep and hard so they can share their expert and wet techniques with their budding blossoms of hotness. Naughty daughters and rivals, beautiful Jesse Jane®, Riley Steele™, Kayden
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30 Free Folf Courses near You
Showing Folf Courses within 50 miles Distance miles of Baltimore, MD, USA City, Zip, or Address
Lake Montebello Disc Golf Course2.9 miles NE
Beautiful views playing around Lake Montebello. Multiple tees/pins with a 6 hole beginner course on the opposite side of the lake. There is a water fountain near the pump house - close to #17 by the...Disc Golf
Lake Montebello - Youth Disc Golf Course2.9 miles NE
At beautiful Lake Montebello. This short recreational course is on the south side of the lake - the opposite side from the 18 hole main course. Public (No Fee)Disc Golf
Druid Hill - Forest Drive3.3 miles NW
Previously called the X-Holes, the Forest Drive course is wooded and challenging. These 18 holes can be played in a single loop or combined with the Legacy course for two 18-hole courses. Public (No...Disc Golf
Do you provide recreation that's not free?
Goucher College Disc Golf Course8.3 miles N
Scenic setting in shaded grove outlined by woods. Thorn bushes off fairways. Short but challenging with elevation. Well maintained. Picnic area, bench and trash cans. Public (No Fee)Disc Golf
Silver Creek Disc Golf Course8.6 miles NW
Lovely stream valley setting with many trees. Bring map to help navigate. 7 baskets - dual tees make 14. Public (No Fee)Disc Golf
Pine Grove Middle School9.2 miles NE
Dual tees makes 18. Long, open and short, wooded fairways. Elevation changes; endurance required. Multiple pin placements. Public (No Fee)Disc Golf
Rockburn Branch9.7 miles SW
On 50 scenic acres with elevation and a variety of open and tight holes. Creek and thorns come into play. Doyle house comes into play on #12, 13, 14, and the signature 15th. Public (No Fee)Disc Golf
CAPA Field17.5 miles E
Flat, open 9 hole course located at Aberdeen Proving Ground, Edgewood (ID Required). Public (No Fee)Disc Golf
Leister Park Disc Golf Course24.1 miles NW
Plays through the open and in woods consisting with some moderate elevation along the walking path. Public (No Fee)Disc Golf
Calvert Road Park27.2 miles SW
Classic layout stresses fundamentals. Benches, multiple tees and alt. pins on every hole. Fun for all. Very well-kept park with picnic and play areas. Convenient to Metro and University of Md.,...Disc Golf
Churchville 627.2 miles NE
For beginners. Public (No Fee)Disc Golf
Scarboro Hills28.8 miles NE
Rolling hills played through woods and open fields. Good variety and elevation. Added contact; Jeff Ramer - mcmlxxatverizon.net. Public (No Fee)Disc Golf
Codorus Township Park34.2 miles N
A mix of open and wooded holes. Some baskets on fast hillside locations. Map can help with navigation, although baskets have next tee directional markers on them. Public (No Fee)Disc Golf
Peake Disc Golf36.8 miles SE
Good variety and challenge on beautifully kept grounds. A pond provides challenge on two holes. Very scenic. Public (No Fee)Disc Golf
Codorus State Park - Blue37.7 miles NW
One of a few courses here. Blue (18 plus 9 Gold X) has open and wooded fairways. Red is open with road and water OB. White has challenging tight fairways. Power, finesse, fairway management and shot...Disc Golf
Codorus State Park - Red37.7 miles NW
Red is open with road and water OB. Blue has open and wooded fairways. Gold X has 9 open and wooded holes (next to Blue course). White has challenging tight fairways. Power, finesse, fairway...Disc Golf
Codorus Pines Mini Disc Golf Course37.7 miles NW
Mini Disc Golf Course in an enchanted forest of Blue Spruce right off a prime picnic area. Also three regular disc golf courses on the property with breathtaking views of the lake. Public (No Fee)Disc Golf
McLean Central Park39.1 miles SW
Near tennis and basketball courts. 2 holes play over creeks. 18 other object holes around Community Center. #4 is a tough birdie!. Public (No Fee)Disc Golf
Darnestown Swim and Racquet Club39.6 miles W
This is a private swim and tennis club. Non-members are welcome to play as long as they respect the club and the members - especially during the summer. This long, technical, woods course uses the...Disc Golf
Clearview Elementary School Disc Golf40.2 miles N
5 baskets surrounding the playground area of an elementary school. Practice basket to basket. Only available for public play outside of school hours. Private (No Fee)Disc Golf


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Has anyone visited this "luxury" outlet mall? If so, do you think it's worth a big detour to go there? The bargain huntress in me is saying, "Si! Si!" but I'd hate to loose 2 days if it's a dud. Also, how far is it from Nice?
Any help would be appreciated!
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Italy-Shopping Outlet Serravalle
Has anyone visited this "luxury" outlet mall? If so, do you think it's worth a big detour to go there? The bargain huntress in me is saying, "Si! Si!" but I'd hate to loose 2 days if it's a dud. Also, how far is it from Nice?
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I am going to Piedmonte in 2 weeks and plan to go shopping at Serravalle. Official sale months in Europe are January and July, and that includes the outlet shops also. Serravalle was the first outlet center developed in Europe and is purported to be very popular, especially on the weekends. That is why we are planning to go on a thursday. In spite of the weakness of the U.S. $, the prices are good because designer goods are cheaper in Italy, in the first place. Since our cottage/apartment is only about 45 minutes drive, we're definitely planning to go, so I can let you know what I think when I return the first part of February. By the way, I think it will be quite a drive from Nice, but check it out on www.mappy.com
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Hi mcguire, rivoli posted August 2003.
But do let us know about your experience in Serravalle, I am sure a lot of people will be interested. Have fun and best wishes that you find some great items!
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I know the OP was in 2003, but for others who may be interested. We were at these outlets this past June. They have a lot of designer stores there, as you can see from their website. However, we did not find any deals, and my daughter and I are major shoppers!! She did find a bathing suit at the Dolce & Gabbana store that wasn't too badly priced (but I decided it was a bit skimpy for her!!) I would not recommend anyone go too far out of your way to stop there, but if you are not too far away and you enjoy shopping, it's probably worth it.
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I was at Serravalle in July. It is ok, but not worth going out of your way for. Conversely, the outlet stores in Valdarno (outside of Firenza) are fabulous. All high end, with incredible deals.
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I thought this thread may be worthwhile bringing back as we will visit Rome again soon. A few years ago I visited the Rome outlet which is near Fiumicino Aiport and Ostia. There I purchased Frette sheets and various pieces of clothing at very attractive prices (particularly when I compared the products and prices in Rome) . I was buying that seasons stock and not old superceeded stock. The only frustration was the rather remote location although they do have a bus to and from Rome with a limited timetable. Has anyone been there in the last few years with any comments on shopping and/or transport to and from there ?
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