Altered expressions of CXCR4 and CD26 on T-helper lymphocytes in hereditary hemorrhagic telangiectasia

Hereditary hemorrhagic telangiectasia (HHT) is a vascular genetic disease with an estimated prevalence of 1 in 5000–8000. Mutations in several genes of the BMP9/BMP10 signaling pathway (ENG and ACVRL1 mainly) result in a deregulated neo-angiogenesis. The inheritance pattern is autosomal dominant, and the penetrance is usually complete after 50 years. The clinical presentation includes spontaneous and recurrent epistaxis (frequently complicated by iron-deficiency anemia), muco-cutaneous telangiectases (face, oral cavity, hands), and less frequently visceral arteriovenous malformations (AVM) of the lungs, the liver, the digestive tract or the central nervous system [1].

Besides this vascular phenotype, some studies reported atypical and severe infections, including cerebral abscesses and musculoskeletal infections, affecting up to 15% of the patients. The actual prevalence and clinical significance of these manifestations have yet to be confirmed by large prospective studies comparing this risk with that of the general population. The most specific events are cerebral abscesses with anaerobic bacteria, generally attributed to septic emboli allowed by right-to-left cardiac shunting due to pulmonary AVM. Patients also seem to be concerned more frequently by musculoskeletal infections involving Staphylococcus aureus, which may be associated with bacteremia provoked by prolonged nose packing or/and lesions of the nasal mucosa [2, 3]. In addition to these mechanical factors, innate immunity is suspected to be altered in HHT: functional deficits of neutrophils and monocytes/macrophages (phagocytosis, oxidative burst, NETs formation) have been reported in humans [4, 5] and mice [6], but their exact clinical meaningfulness as well as their underlying mechanism are still to be elucidated. Intriguingly, immunological studies have highlighted a T and NK lymphopenia predominant on naive T-helper cells [5, 7]. Although very low lymphocyte counts were observed in some patients, no clear association with infectious risk has been established to date.

The chemokine CXCL12 (formerly named SDF1) and its cellular receptor CXCR4 constitute a powerful chemotactic axis for many cell types. It is in particular used by T lymphocytes for trafficking and homeostasis [8]. CD26 is a known CXCL12 inactivating peptidase, functionally associated with CXCR4 on T lymphocytes [9]. Besides this enzymatic activity, CD26 acts as a co-stimulatory molecule during T-lymphocyte activation [10, 11]. In endothelial cells, CXCL12 and CXCR4 expressions are regulated by BMP9 in an endoglin-dependent manner: BMP9 and hypoxia are additive inducers of CXCL12 release while surface CXCR4 is downregulated [12]. Imbalance of this chemotactic axis has also been described on peripheral blood mononuclear cells (MNCs) in one study. The migration capacities of HHT MNCs towards a CXCL12 gradient were reduced as a result of a CD26 hyper-expression, which was overcoming elevated surface levels of CXCR4 [13].

Here, we describe the CXCR4 and CD26 expression levels in different lymphocyte subsets of HHT patients with history of severe infection (HSI), in comparison with HHT patients without HSI and with healthy control subjects (HC) matched in age and sex. We provide several elements supporting an alteration in the CXCL12/CXCR4 axis on T-helper lymphocytes in HHT patients, possibly related to their specific infectious risk and T-helper lymphopenia.

CXCR4 is the G protein-coupled receptor of the pleiotropic chemokine CXCL12. This chemotactic axis is fundamental during organogenesis, angiogenesis and homeostasis of the hematopoietic and immune systems. CXCL12 is highly expressed in bone marrow and is locally upregulated via HIF-1 after tissue injury to recruit stem cells and progenitors for damage repair [15, 16]. On T lymphocytes, CXCR4 is co-localized with CD26 (dipeptidylpeptidase IV), and both proteins are co-internalized after activation by CXCL12. They form a functional unit participating in chemotaxis towards bone marrow and lymph nodes [9]. This pathway is involved in several diseases, including cancer [17], rheumatoid arthritis [18] and HIV [19]. Here, we report for the first time elements supporting a dysregulation of this axis on T lymphocytes in HHT.

As already published [5, 7], HHT is associated with a T and NK lymphopenia despite the absence of any comorbidities or treatment known to cause lymphopenia [20]. The decrease of the T-helpers could be a consequence of a dysfunctional CXCL12/CXCR4 axis since we observed a weak but significant correlation between their CXCR4 surface expression and their absolute counts in the blood. This hypothesis is reinforced by the absence of such correlation in the control group. This phenomenon could be due to a direct effect of CXCL12 on naive T lymphocyte survival [21] or through disturbance in the T lymphocyte recirculation in lymph nodes and bone marrow, in which the CXCR4/CXCL12 axis is known to be important [8, 22]. The human aging of the immune system seems associated with a deregulation of CXCR4 expression on T-helper cell surface [23], but a such phenomenon is not observed in our study. The association between iron treatment and T-helper lymphopenia is more difficult to explain. It could be due to a direct effect of the treatment, maybe by increasing the oxygen reactive species in peripheral blood [24]. It also could be a consequence of the iron deficiency, which is known to induce a reduction in peripheral T cells and atrophy of the thymus [25]. As a third hypothesis, there could exist a confounding factor, such as a subpopulation of patients with a more strongly activated neo-angiogenesis, impacting on both iron requirements and lymphocyte recruitment. Interventional studies are necessary to differentiate these hypotheses, especially since a link between the risk of cerebral abscess and IV iron loading has been reported [26].

We observed a correlated decrease in CXCR4 and CD26 expressions on T-helper lymphocytes of HHT patients without modification of the CXCR4/CD26 ratio. This is in apparent contradiction with a previous report of an over-expression of CXCR4 and CD26 on mononuclear cells with a decreased CXCR4/CD26 ratio associated with chemotactic impairment [13]. This discrepancy could be due to a different pattern of CXCR4 and CD26 surface expression between lymphocytes and monocytes. The monocytes have never been specifically studied in human HHT but are known to express these markers [27, 28]. Moreover, the previous study included only ENG-mutated patients. On T-helper lymphocytes, CXCR4 under-expression seems to be a central phenomenon in the idiopathic CD4 lymphopenia [29], but the infectious profiles are quite different. An alternative explanation could be a chronic elevation of the CXCL12 plasma level, described in severe infection [30, 31] and recently in HHT [32]. Such an elevation is known to lead to continuous CXCR4 and CD26 internalization as a desensitization process [9, 33]. The under-expression of CXCR4 on T-helper lymphocytes could delay and mitigate their antigenic response by limiting their migration and activation capacities [34‐36]. The decrease of CXCR4 and CD26 surface expression seems to spare the T-cytotoxic and the NK lymphocytes. This observation is surprising since both molecules have documented biological roles on these cell types [11, 35, 37‐39]. It could result from their lower basal expression levels, limiting our ability to distinguish small variations.

We describe for the first time a significantly higher CXCR4/CD26 ratio on naive T-helper lymphocytes of HHT patients that have experienced at least one serious infectious event. The magnitude of this effect on clinical risk will require confirmation by further studies and may not be as large as the effect of pulmonary AVMs, the risk factor well described in HHT [2] and confirmed here. Since CXCR4 and CD26 are functionally linked, it is logical to suspect a higher chemotaxis of naive T-helper lymphocytes in HHT patients with HSI. This can appear paradoxical in an immunodeficient state, but such a phenomenon, extended to the whole immune system, has a central role in the physiopathology of the WHIM syndrome [40]. In this disease, most of the infectious phenotype seems to be due to the chemotactic dysfunction of the myeloid lineage, but patients also exhibit a decrease of NK and naive T lymphocytes [41]. However, the validity of a similar paradoxical mechanism underlying HHT-related infection profile is not obvious, since WHIM syndrome is due to a CXCR4 gain-of-function mutation, resulting in prolonged retention of leukocytes in bone marrow and, consequently, panleukopenia, whereas no evidence of such behaviour is currently available for naive T-helper lymphocytes in HHT patients. Of note, the CD26 expression on T lymphocytes is regulated upon activation in inflammatory contexts such as rheumatoid arthritis or allograft rejection [11, 42]. Therefore, inflammation could also participate in the shift of the CXCR4/26 ratio in HHT patients with a history of severe infection, despite the significant time elapsed between the last episode of infection and the time of enrollment.

Our study suffered from several limitations. The first is the low number of subjects, limiting the statistical analyses. This fact is inherent to studies on rare diseases. Nevertheless, the ability to identify pulmonary AVM as an infectious risk in HHT allows us to think that our population size was large enough to spot clinically relevant parameters. Another limitation comes from the absence of data about the lymphocyte profiles prior to the infectious episodes. We do not provide any proof that the modified expressions of CXCR4 and CD26 are stable over time, knowing that lymphocyte profiles have circadian and seasonal variations [43‐45]. Nevertheless, the major lymphocyte subsets of a given individual have been reported with a low level of variability over time [46‐48]. The assessment of CXCR4 and CD26 expression is only based on flow-cytometry data and should be confirmed by other techniques (RNA quantification, for example). Plasma levels of chemotactic agents (especially CXCL12) and functional assessment of T lymphocyte chemotaxis could have brought more interpretability in our results. These investigations should be conducted in future studies on this topic, as well as a more detailed evaluation of the Th1, Th2 and Th17 lymphocyte sub-populations.

We report several findings suggesting that HHT is associated with an alteration of the CXCL12/CXCR4 chemotactic axis of T-helper lymphocytes. Patients exhibit a decrease of their total count in the blood correlated with a decrease of CXCR4 MFI ratio, a decrease of CXCR4 and CD26 surface expressions and a higher CXCR4/CD26 ratio on the naive subset in the group with a history of severe infection. As the CXCL12/CXCR4 chemotactic axis is important in many parts of the immune system, it may contribute to the greater susceptibility to infection observed in HHT.