Numerous studies have demonstrated the importance of HO-1 and its enzymatic products as anti-inflammatory mediators in various disease states (12
). Compared to wild-type mice, HO-1 knockout mice develop a progressive inflammatory state and splenocytes from these mice respond to TCR activation with increased production of pro-inflammatory cytokines such as IL-2, IFN-γ, TNF-α, GM-CSF, and IL-6 (16
). Such effects have been observed in human cells as well, where HO-1 and CO inhibit T cell proliferation in response to activation through the TCR in vitro
). Furthermore, HO-1 activity in APCs such as dendritic cells (DCs) and cells of the monocyte/macrophage lineage can significantly influence the outcome of their interactions with T cells (25
). For example, splenocytes from HO-1 knockout mice display enhanced production of IL-6, TNF-α, IFN-γ, and IL-12 in response to LPS stimulation ex vivo
). Chauveau et al
. showed that induction of HO-1 expression in rat and human DCs led to impaired LPS-induced activation and maturation, and impaired ability to stimulate allogeneic T cell proliferation (25
). Recent work by Tzima et al
has also demonstrated a role for myeloid-expressed HO-1 in triggering innate immunity (35
): mice carrying a myeloid-specific ablation of the HO-1 gene had impaired production of IFN-β in response to viral and bacterial infection, and a more severe disease course after induction of experimental autoimmune encephalomyelitis. Together, these results suggest that HO-1 in myeloid cells may play a complex and important role in T cell activation and differentiation. Given that HO-1 over-expression and CO exposure can inhibit T cell activation via the TCR (11
), and that HO-1 can inhibit T cell activation by APCs (25
), we considered whether HO-1 might exert some function at baseline in maintaining T cell quiescence. Specifically, we asked whether exposure to the potent pharmacologic HO-1 inhibitor, SnMP, would result in T cell activation.
Here, we demonstrate that the HO-1 inhibitor, SnMP, induces activation, proliferation, and maturation of naïve CD4+ and CD8+ T cells via interactions with CD14+ monocytes in vitro. Notably, SnMP did not induce proliferation in isolated T cells, but only in cultures where CD14+ cells were also present. While this observation does not rule out a direct effect of HO-1 on T cells, it does indicate that such an effect is not sufficient to induce activation. Proliferation occurred in the presence of very few monocytes, and there was a direct correlation between the frequency of monocytes present in culture and the extent of proliferation. Experiments using transwells and blocking antibodies demonstrated that SnMP-induced T cell activation requires direct cell-to-cell MHC Class I and II-dependent interactions between T cells and monocytes. Both MHC Class I and II blockade inhibited SnMP-induced proliferation, and there was an amplified effect of dual blockade, with abrogation of proliferation even at low antibody concentrations.
Given that MHC-dependent interaction of monocytes with T cells plays a crucial role in this in vitro system, we analyzed the phenotypic changes that occur in CD14+ cells on day 3 of culture, prior to observed T cell proliferation. In the absence of additional cytokines, monocytes in PBMC culture normally stick to plastic plates and differentiate into monocyte-derived macrophages (MDM), which we see occurring in vehicle control samples, where CD14+ cells are also CD11c+, CD16+, and HLA-DR+. We noted several differences in CD14+ cells that were cultured in SnMP. Among the effects noted were a decrease in the expression of both CD11c and CD16. Most notably, SnMP induced upregulation of the co-activating molecule CD86 (B7-2), which plays an important role in the MHC-TCR immunological synapse by providing crucial secondary signals that modulate T cell activation. We postulate that this upregulation may enhance the ability of monocyte-derived CD14+ cells to activate T cells via TCR-MHC interactions. The C-type lectin BDCA-2, which is typically expressed on plasmacytoid dendritic cells, was also significantly upregulated in SnMP-treated CD14+ cells. Together, these changes demonstrate that the CD14+ population undergoes several phenotypic changes in response to HO-1 inhibitor exposure, some of which have the potential to confer activating function, while HO-1 induction by CoPP is associated with changes that are associated with a non-inflammatory phenotype (i.e., a decrease in the expression of CD86 and HLA-DR).
We found that SnMP-induced T cell proliferation can be inhibited by CD25+
Tregs but that, reciprocally, SnMP can inhibit the suppressive function of Tregs. Tregs from HO-1 deficient mice have no intrinsic defect in their ability to suppress T cell activation, but it is now known that their ability to do so maximally and efficiently requires interactions with wild-type APCs that have intact HO-1 activity (26
). Accordingly, we suggest that inhibition of HO-1 activity in APCs in human PBMC cultures results in impaired Treg function. It is widely accepted that Tregs can induce changes in APCs to down-regulate their antigen presenting functions (37
Conversely, both immature DCs and alternatively activated macrophages can induce Treg development de novo
, while classically activated macrophages and mature dendritic cells can have negative effects on Treg function (39
). The mechanism by which HO-1 activity supports Treg function remains a matter for speculation. CO produced by HO-1 in APCs could act in a paracrine fashion to support Tregs by suppressing T cell proliferation or by inducing transcriptional changes in the Tregs themselves, leading to enhanced survival or suppressor activity. Catabolic products of heme breakdown could also work in an autocrine fashion to drive APC differentiation toward a phenotype that supports Treg survival or function.
Based on the results of our experiments, we suggest the model shown in to describe the interactions leading to T cell activation and proliferation in PBMC cultures upon HO-1 inhibition by SnMP. In this model, unopposed baseline endogenous HO-1 activity supports the quiescent state of monocytes. There may be an endogenous effect of HO-1 in both naive T cells and Tregs, but it is also likely that the effects of HO-1 are exerted via interactions with quiescent monocytes that promote Treg survival and function. In this baseline state, anti-proliferative signals prevail, and interaction with self-MHC allows for T cell survival and low-level baseline rates of proliferation. Exposure to SnMP results in HO-1 inhibition, leading to pro-activating phenotypic changes in monocytes, naïve T cells, or both. The primary observed effect resulting from this is the MHC-dependent induction of T cell proliferation. HO-1 inhibition also results in monocyte-mediated impairment of Treg function, indirectly augmenting the extent of naïve T cell proliferation. Together, these effects are sufficient to induce proliferation of a surprisingly large fraction of T cells present in PBMC cultures.
Figure 6 Model of SnMP-induced T cell activation and proliferation. (A) At baseline, active HO-1 in monocytes (Mono) exerts an inhibitory influence on CD45RA+CD27+ naïve T cells (TN) and simultaneously supports function of CD4+CD25+FoxP3+ regulatory T (more ...)
While we base our model on the assumption that the enzymatic activity of HO-1 is responsible for the observed effect, it is important to consider the alternative possibility that non-enzymatic functions of HO-1 play a role. Recent work has shown that HO-1 possesses important transcriptional modifying activity that is completely independent of its catalytic function. In NIH 3T3 cells exposed to hypoxia or heme, HO-1 underwent cleavage of a C-terminal domain, leading to nuclear translocation of the N-terminal domain of HO-1 and subsequent transcriptional regulation by this cleaved portion (40
). Furthermore, HO-1 protein that has been made to be catalytically inactive through site-directed mutagenesis participates directly in its own transcriptional autoregulation despite the absence of an active catalytic site (41
). Thus, the phenotypic changes observed in response to SnMP may also be related to transcriptional changes induced by the presence of non-catalytically active (i.e. inhibited) HO-1 protein. This possibility is especially intriguing since HO-1 expression is induced by SnMP, resulting in a relative excess of inhibited HO-1. Of note, we attempted to carry out spectrophotometric HO-1 enzyme activity assays to confirm induction and inhibition of HO-1 (data not shown), but were limited by the number of cells available from an individual donor. Normally this assay is carried out on tissue or cell-line lysates, from which protein yield is not normally limiting. We were unable to generate sufficient quantities of microsomal protein from single volunteer human donors to carry out this assay successfully, and so were unable to directly demonstrate that SnMP inhibits HO-1 activity in our system. There is ample evidence from the literature that synthetic metalloporphyrin inducers and inhibitors of HO-1 are active in hematopoetic cells, and specifically in cells of the monocyte/macrophage lineage (42
), and so it is reasonable to assume that SnMP acts as an inhibitor in our system.
The findings of this in vitro
study suggest that HO-1 plays a role in controlling naïve T cell activation, maturation, and proliferation, and in vivo
studies are clearly warranted to validate the physiological relevance of our findings. The goal of such studies would be to determine if HO-1 activity represents a safeguard mechanism to prevent non-specific T cell activation by APCs, and whether removal of this safeguard by HO-1 down-regulation or inhibition plays a role in promoting T-cell activation and maturation under physiologic or pathologic circumstances. Activation of T cells in vitro
by SnMP required interaction with MHC Class I and II, presumably via the TCRs on responding T cells. This is notable in that the observed response almost certainly represents a TCR-mediated response to self-MHC. Normally, T cells do not undergo widespread activation and proliferation in response to self-MHC, which is crucial for the maintenance of tolerance to self and prevention of autoimmunity. There are many mechanisms in place to ensure that T cell activation occurs only in appropriate settings (e.g., upon exposure to dangerous pathogens or upon detection of malignancy) and not in response to self-antigens. Chief among these mechanisms is the thymic deletion of autoreactive T cells through negative selection (45
) and, in the peripheral immune system, the maintenance of tolerance by regulatory cells such as CD4+
). These regulatory cells also participate in the tuning and modulation of immune responses to ensure their appropriate activation and termination. In the absence or relative paucity of these regulatory mechanisms, the immune response may proceed unchecked, causing collateral damage to the host (46
). Given the extent of proliferation observed after HO-1 inhibitor exposure, we posit that HO-1 may also serve as a safeguard mechanism to prevent inappropriate T-cell activation. In many animal disease models, absence of HO-1 activity results in excess inflammation that contributes to pathology, including models of diabetes, asthma, multiple sclerosis, cerebral malaria, and transplant rejection (12
). The work presented here provides further support for a potential role of HO-1 in preventing inappropriate T cell activation in humans.
Naïve T cells in the periphery undergo low levels of homeostatic proliferation until they encounter cognate antigen in the context of activating signal, at which point they go on to become effector and memory cells (48
). This homeostatic proliferation is now thought to occur almost exclusively in lymph nodes, where T cells move through the parenchyma and come into contact with fibroblastic reticular cells (49
). Among the signals that are crucial for naïve T cell survival and proliferation, one of the most important is contact with MHC molecules on supporting accessory cells (48
). In addition to the influence of critical growth factors, low-avidity interactions between the TCR on naïve T cells and MHC provide survival signals that allow these cells to continue to proliferate at low levels, thereby maintaining a diverse and appropriately quiescent naïve T cell population. Our experiments suggest that myeloid HO-1 activity may represent a “braking” mechanism for naïve T cell proliferation, allowing for low-level proliferation in response to self-MHC while preventing uncontrolled activation and proliferation. If so, its absence could lead to dysregulated homeostasis. Indeed, mice that are deficient in HO-1 have clear evidence of dysfunctional lymphocyte homeostasis, including splenomegaly, lymphadenopathy, altered CD4/CD8 ratio, and disorganized lymph node and splenic architecture (14
). They also have a higher frequency of activated T cells (15
). This suggests that HO-1 plays a role in the regulation and maintenance of the peripheral T cell pool, and/or in the prevention of inappropriate activation.
Our study suggests that HO-1 plays a role in T cell homeostasis, and support for this hypothesis is found most convincingly in our examination of the maturational profile of cells treated with SnMP. The experiments shown in clearly demonstrate that proliferating cells are primarily naïve T cells that adopt memory cell phenotypes, a phenomenon which is observed in some models of homeostatic T cell proliferation (50
). For example, naive T cells transferred into syngeneic lymphopenic hosts repopulate the host’s peripheral immune system by undergoing robust self-MHC dependent proliferation, during which they take on the phenotype and characteristics of memory cells (50
). It may be that the naïve cells that become proliferating memory cells in our experiments are undergoing a similar homeostatic proliferative response. It remains to be seen whether comparable responses may occur in vivo
during inhibition of HO-1. Certainly, this type of response would seem more likely to occur in secondary lymphoid organs, where T cells come in contact with myeloid cells for an extended period of time. Furthermore, variations in HO-1 activity that would theoretically lead to more or less T cell proliferation could occur in specialized subanatomic regions, possibly influenced by concentration gradients of natural HO-1 inducers such as heme.
HO-1 has been shown in many instances to be anti-proliferative and to down-regulate potentially harmful inflammatory responses. The experiments presented here raise another possible role for HO-1 in T cell homeostasis. Namely, that unopposed HO-1 in myeloid cells provides homeostatic signals to T cells, allowing them to remain in a non-activated state. In the absence of HO-1, or in the presence of inhibited HO-1, a different set of signals (or perhaps merely the absence of anti-proliferative signals) may then lead to T cell activation and proliferation. This effect may also represent a mechanism to alleviate suppression of T cells in settings where activation is needed, such as infection or malignancy. In aggregate, these findings demonstrate that HO-1 can alter human T cell activation, maturation, and proliferation in vitro, and suggest that this multifunctional protein may play a role in controlling lymphoid development and homeostasis in vivo. They also suggest the possibility that SnMP, or other pharmacologic HO-1 inhibitors, could be used as clinical modulators of T cell maturation, which would have potential use in settings requiring immune reconstitution such as chemotherapy and following initiation of highly active antiretroviral therapy for HIV.