Thalassemia carriers exhibit reduced expression of the receptor for the malarial parasite Plasmodium vivax on erythrocytes Open Access

TO THE EDITOR:

Thalassemia, an inherited blood disorder caused by mutations in α-globin or β-globin gene clusters, is characterized by microcytic hypochromic anemia and presents a wide spectrum of clinical severity.¹ The high prevalence of thalassemia in malaria-endemic regions, such as the Mediterranean basin, the Middle East, tropical and subtropical Africa, and the Asian subcontinent, suggests that thalassemias may have been selected to their current frequencies through a survival advantage in malaria-endemic environments.² Several mechanisms underlying this protective effect have been proposed, including increased microerythrocyte counts, reduced parasite growth, enhanced clearance of infected cells, and altered erythrocyte environments.^3,4 However, the complete mechanisms that confer resistance to malaria remain elusive.

Analysis of the species composition of malaria cases reveals that Plasmodium falciparum is the dominant species in Africa, whereas Plasmodium vivax is the most prevalent species in Asia, Oceania, and large parts of the Americas.⁵ Interestingly, the highest prevalence of thalassemia is in regions endemic for P vivax malaria.^5-7 CD234, an erythrocyte receptor for P vivax,⁸ is also known as the Duffy antigen/receptor for chemokines which serves as a regulatory component of chemokine networks under both physiological and pathological conditions.⁹ Here, we hypothesize that thalassemia carriers exhibit decreased levels of CD234 and this reduction may not only interfere with the invasion of erythrocytes by P vivax but also regulate immune responses directed toward the parasites.

In this study, we aim to determine the expression levels of CD234 and the profiles of inflammatory chemokines in thalassemia carriers. For this purpose, 83 female thalassemia carriers and 73 sex-matched healthy controls were recruited in this study from the Hainan Women and Children’s Medical Center (HNWCMC), Haikou, China, between July and August 2023 (supplemental Methods).

As summarized in supplemental Table 1, the average age of thalassemia carriers and control subjects was 33.6 and 30.5 years, respectively. The thalassemia group consisted of 32 α-silent, 35 α-thalassemia minor, 9 β-thalassemia minor, and 7 β-thalassemia minor combined with α-silent or α-thalassemia minor carriers. Compared with healthy controls, thalassemia carriers had higher red blood cell counts and lower levels of hemoglobin concentration, mean corpuscular volume and mean corpuscular hemoglobin. The genotypes of all 83 thalassemia carriers are summarized in supplemental Table 2.

The mean fluorescence intensity (MFI) of CD234 in healthy controls was 3169 ± 477, which was substantially higher than that in thalassemia carriers (2480 ± 532; P < .0001) (Figure 1A). The MFI of CD234 in α-silent, α-thalassemia minor, β-thalassemia minor, and β-thalassemia minor combined with α-silent or α-thalassemia minor carriers was 2788 ± 490, 2273 ± 454, 2069 ± 382, and 2635 ± 488, respectively, all of which were significantly lower than those in controls (Figure 1B). After normalizing CD234 levels to cell surface area, a significant decrease in CD234 expression was still observed in all thalassemia carriers (P < .0001), including those with α-thalassemia minor (P < .0001) and β-thalassemia minor (P < .001), when compared with healthy controls (Figure 1C-D).

We also determined the expression of CD235a, a receptor for P falciparum.⁸ The MFI of CD235a in healthy controls was slightly higher than that in thalassemia carriers (18885 ± 3701 vs 16772 ± 3609; P < .001) (Figure 1E). Regarding subgroups of thalassemia carriers, both α-thalassemia minor carriers had significantly decreased levels of CD235a compared with healthy controls (P < .0001) (Figure 1F). However, after normalizing CD235a levels to cell surface area, thalassemia carriers exhibited comparable CD235a expression levels to those of healthy individuals (Figure 1G-H).

Given that CD234 is an atypical chemokine receptor that binds both CC and CXC chemokines and regulates their activity,⁹ we next determined the serum profile of 13 inflammatory chemokines in thalassemia carriers and controls. Among the 13 tested chemokines, levels of CCL2 (38.5 pg/mL vs 51.0 pg/mL; P < .01), CCL11 (26.0 pg/mL vs 32.5 pg/mL; P < .01), and CCL17 (9.5 pg/mL vs 13.6 pg/mL; P < .05) were lower in thalassemia carriers than in healthy controls, whereas levels of CXCL11 were higher in thalassemia carriers than in healthy controls (41.6 pg/mL vs 36.0 pg/mL; P < .05) (Table 1). Similar results were observed in subgroups of thalassemia carriers. Compared with healthy control subjects, α-thalassemia minor carriers had lower levels of CCL2, CCL11, and CCL17, and β-thalassemia minor carriers had increased levels of CXCL11 (Table 1).

Due to the microcytic nature of thalassemia, it is expected that the levels of CD235a and CD234 on erythrocytes are lower in thalassemia carriers than in healthy controls. However, it is notable that the decrease in CD234 levels is more pronounced than that in CD235a levels. This difference becomes particularly evident when the levels of the 2 molecules are normalized to the cell surface area. Although the normalized levels of CD234 were significantly lower in thalassemia carriers compared with healthy controls, the normalized levels of CD235a were comparable between the 2 groups. This suggests a specific decrease in CD234 expression on erythrocytes in thalassemia carriers.

In 1975, Miller et al reported that individuals with a Duffy blood group–negative genotype are resistant to infection by P vivax,¹⁰ revealing CD234 is the receptor for the parasite. Although recent studies have reported P vivax infections in Duffy-negative individuals across Africa, this can be explained by the fact that the globin transcription factor 1 polymorphism does not completely abolish Duffy expression.^11,12 Furthermore, treatment with chymotrypsin, which leads to the shedding of CD234 from the erythrocyte surface, inhibits the invasion of CD234-positive human erythrocytes by Plasmodium knowlesi, a malaria parasite of monkeys, in a dose-dependent manner,¹³ suggesting that levels of CD234 on erythrocytes correlate with malaria pathogen invasion. Therefore, the observed decreased levels of CD234 on erythrocytes in thalassemia carriers imply that thalassemia heterozygosity might confer resistance to vivax malaria infection by limiting the red blood cell surface expression of CD234.

Interestingly, serum levels of several inflammatory chemokines that can bind CD234, namely CCL2, CCL11, CCL17, and CXCL11, were decreased in 1 or more subgroups of thalassemia carriers compared with controls. As key regulators of leukocyte trafficking, chemokines are proposed to play an essential role in the development of severe malaria.¹⁴ The aforementioned 4 CD234-associated chemokines have been implicated in malaria infection or its animal models. For instance, CCL2, also known as monocyte chemotactic protein 1, has been suggested as a biomarker for severity in P vivax malaria.¹⁵ In a mouse model of cerebral malaria, CCL17 is identified as a crucial factor for enhanced survival in the absence of cannabinoid receptor 2,¹⁶ and levels of CCL11 are elevated in the hippocampus, associated with impaired neurogenesis and cognitive/memory impairment.¹⁷ Therefore, it is conceivable that the decreased levels of these 4 circulating chemokines in thalassemia carriers might influence the progression of malaria infection. However, this hypothesis requires experimental validation. Furthermore, given that these chemokines are not exclusively associated with the immune response against malaria, their decreased levels could potentially contribute to susceptibility to other infectious diseases. This notion is supported by previous observations indicating that α-thalassemia confers protection not only against malaria but also against other infections.¹⁸ 

This study has 2 notable limitations. First, the cell surface area used to normalize the levels of CD234 and CD235a was calculated under the assumption that erythrocytes are spherical. However, because typical erythrocytes are biconcave in shape, this approximation introduces inaccuracies in the calculated surface area, potentially compromising the results. Second, the levels of CD234 were quantified using a specific monoclonal antibody. The epitope specificity and binding affinity of the antibody may influence the quantification. Therefore, it would be valuable to validate this finding using an alternative monoclonal antibody against CD234.

In summary, this study demonstrates that thalassemia carriers exhibit substantially decreased levels of CD234 on the surface of erythrocytes and lower serum levels of multiple CD234-bound chemokines, which have been associated with malaria infection. These findings implicate that thalassemia heterozygosity may confer protection against P vivax through the regulation of CD234 and inflammatory chemokines. It is important to investigate whether this potential mechanism is also present in other hemoglobinopathies, where carrier status confers resistance to malaria.

This study was conducted in accordance with the principles of the Declaration of Helsinki. Approval for the study protocol was obtained from the Ethics Committee of the HNWCMC (Approval No.: HNWCMC-2023-80). Written inform consent was obtained from all participants in the study.

Acknowledgments: The authors deeply thank the patient and her family for supporting this research.

This work was funded by the Natural Science Foundation of Hainan Province (823QN359), the Major Science and Technology Project of Hainan Province (ZDKJ2021037), Hainan Province Clinical Medical Center (QWYH202175), the Excellent Talent Team of Hainan Province (QRCBT202121), and the German Ministry of Education and Research through the German Center for Lung Research (DZL4.0).

Contribution: X.Y. and Q.Z. designed and supervised the research; R.H., Xinze Li, Z.L., J.X., D.L., Y.F., Y.L., and Xuexia Li performed the research; R.H. analyzed and interpreted the data; and X.Y., R.H., and F.P. wrote and revised the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Xinhua Yu, Priority Research Area Chronic Lung Diseases, Research Center Borstel, Parkallee 22, 23845 Borstel, Germany; email: xinhuayu@fz-borstel.de; and Qiaomiao Zhou, Hainan Women and Children's Medical Center, Longkunnan Rd 75, 571199 Haikou, China; email: zhouqiaomiao@163.com.