REVIEW URRENT C OPINION
Neutrophil counts in persons of African origin Christina F. Thobakgale a,b,c and Thumbi Ndung’u a,b,c,d
Purpose of review The causes of ethnic or benign neutropenia have long been unclear. Here, we discuss the emerging data on the causes and consequences of neutropenia and discuss the relevance of these data for African populations, in which the prevalence of neutropenia is high. Recent findings Genetic deletion of the Duffy antigen receptor for chemokines (DARC-null genotype) has been identified as a major determinant for neutropenia. DARC acts as a receptor for Plasmodium vivax malaria and the DARC-null genotype has thus been positively selected among Africans; however, recent studies suggest that Duffy-null-linked neutropenia could increase the risk of HIV infection. Data are emerging that neutrophils are versatile cells that play a critical role not only in direct antimicrobial activity but also in priming and regulating the activity of other innate and adaptive immune cells. Therefore, we discuss here the imperative to better understand the causes, consequences, and the underlying mechanisms of neutropenia among Africans as a prerequisite for rational and optimal biomedical interventions to improve health outcomes. Summary Neutropenia among Africans, linked to the Duffy-null trait or otherwise, may have significant health consequences that remain largely undetermined and could have a significant impact on the pathogenesis of diseases. Keywords Africans, antimicrobial immunity, Duffy-null, neutrophils, phenotype
INTRODUCTION Neutrophils have emerged as versatile cells capable of direct antimicrobial activity and immune regulation. Thus, a reduction in their numbers as seen in rare cases of severe congenital neutropenia can have life-threatening implications. In contrast, ethnic or benign neutropenia is common among Africans and is thought to be of no adverse clinical impact. In this review, we address the recent findings demonstrating that neutrophils play a critical role in controlling infection and inflammation. We discuss data on the genetic basis of congenital and benign neutropenia and association of neutropenia with infection and disease outcomes. We also highlight that there is paucity of data on the causes and implication of benign neutropenia on disease and propose that epidemiological, genetic, and mechanistic studies will be required to unravel whether this condition is detrimental or beneficial in specific circumstances. This research is needed to optimize interventions and improve health outcomes in African ancestry populations. www.co-hematology.com
NEUTROPHILS ON THE FRONT LINE AND AS REGULATORS OF ADAPTIVE IMMUNITY Neutrophils, also known as polymorphonuclear leukocytes (PMNs), are innate immune cells, approximately 7–10 mm in diameter, and characterized morphologically by the presence of a segmented nucleus and cytoplasmic granules and secretory a
HIV Pathogenesis Programme, Doris Duke Medical Research Institute, KwaZulu-Natal Research Institute for Tuberculosis and HIV, Nelson R. Mandela School of Medicine, University of KwaZulu-Natal, Durban, South Africa, cRagon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology, and Harvard University, Boston, Massachusetts, USA and dMax Planck Institute for Infection Biology, Berlin, Germany b
Correspondence to Thumbi Ndung’u, PhD, HIV Pathogenesis Programme, Doris Duke Medical Research Institute, Nelson R. Mandela School of Medicine, University of KwaZulu-Natal, 719 Umbilo Road, Durban, 4013, South Africa. Tel: +27 31 260 4727; fax: +27 31 260 4623; e-mail: [email protected] Curr Opin Hematol 2014, 21:50–57 DOI:10.1097/MOH.0000000000000007 Volume 21 Number 1 January 2014
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Neutrophils in Africans Thobakgale and Ndung’u
KEY POINTS Neutrophils can inhibit various pathogens directly through phagocytosis, degranulation, and NETosis. Neutrophils also interact with other immune cells such as T cells, B cells, macrophages, natural killer cells, platelets, and dendritic cells. These interactions can shape the subsequent immune outcomes. Benign neutropenia is common among Africans and considering the important role that neutrophils play in disease pathogenesis, this condition could be detrimental or beneficial depending on the particular pathogen and host characteristics. The biological basis and consequences of ethnic neutropenia require further research for improved biomedical interventions.
vesicles. Neutrophils originate from precursor myeloid cells in the bone marrow, where they differentiate into myeloblasts before maturing into neutrophils. With about 1–2 1011 cells produced per day, they are the most abundant white blood cells in humans and constitute about 50–70% of leukocytes in circulation [1], and are classically known for the role they play in acute immune response against bacterial and fungal infections. The chemokine receptor CXCR4 (ligand SDF-1) is crucial for maintaining neutrophils in the bone marrow, whereas CXCR2 (ligands KC and Grob) is responsible for neutrophil release. Granulocyte colony-stimulating factor (G-CSF) stimulates neutrophil release directly and indirectly by reducing SDF-1 expression and enhancing the expression of Grob on endothelial cells [2]. Thus, neutrophil homeostasis is maintained by a fine balance between production, storage, and release of neutrophils from the bone marrow, margination, and clearance [3,4]. Neutrophils engulf and eliminate their pathogens by the mechanisms of phagocytosis, degranulation, or formation of neutrophil extracellular traps (NETs) [5 ,6 ]. Emerging reports over the last few years indicate that neutrophils have immune functions beyond phagocytosis and resolving inflammation. In addition to producing cytokines and chemokines [5 ], neutrophils also engage in bidirectional interactions with other immune cells and are emerging as regulators of the innate and adaptive immune responses. The neutrophil’s interaction with other immune cells, including macrophages [7], dendritic cells [8], natural killer (NK) cells [9], B cells [10], and T lymphocytes [11], has only come to light in recent studies and is still poorly understood; however, &&
&&
&&
these studies highlight the potential importance of neutrophils in regulating innate and adaptive immune responses.
NEUTROPHILS IN DISEASE Given the abundance and important role of neutrophils as the first line of immune defense against pathogens and as regulators of innate and adaptive immunity, it is not surprising that total absence of neutrophils or marked decrease in their numbers results in profound immunodeficiencies. Neutropenia is defined as a reduction in absolute neutrophil counts to below 1500 cells/ml for older children and adults regardless of ethnicity [12]. Neutropenia can be stratified according to the clinical severity with absolute neutrophil counts between 1000 and 1500 cells/ml being defined as mild neutropenia, 500–1000 cells/ml is considered moderate neutropenia, whereas counts below 500 cells/ml is termed severe neutropenia [13]. The risk of neutropeniaassociated disorders greatly increases with absolute neutrophil counts below 500 cells/ml. Acute neutropenia, defined as normally resolvable short-term drop in absolute neutrophil counts because of infection or injury, occurs very frequently compared with congenital or cyclic neutropenia which occur only rarely in the general population at 6.2 cases per million [14]. The biological basis, clinical presentation, and management of congenital and cyclic neutropenic disorders have been discussed in detail by others [15 ,16,17 ]. &&
&
FEATURES AND GENETICS OF ETHNIC BENIGN NEUTROPENIA Neutrophil counts vary greatly by ethnic groups. Low absolute counts are a common occurrence in people of African descent and some ethnic groups from the Middle East, a condition known as ethnic or benign neutropenia. Benign ethnic neutropenia occurs in 25–50% persons of African descent and does not appear to be associated with clinical disadvantage compared with matched population controls [12,18–20]. The condition is diagnosed by repeated low measurements of absolute neutrophil counts less than 1500 cells/ml (usually 1000 cells/ml or below) for several months without identifiable causes of neutropenia. Unlike congenital neutropenia, usually diagnosed among whites and potentially life threatening, ethnic neutropenia is clinically inconsequential as it is not associated with increased risk of mucosal or systemic infections [21]. The reported prevalence of benign neutropenia in different ethnic groups is summarized in Table 1 and includes reports from peoples of African descent,
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Myeloid biology Table 1. Prevalence of benign neutropenia by ethnic populations Ethnic group
Country of origin
Benign neutropenia prevalence (%)
Reference
American Blacks
United States
4.5
[19]
American Blacks
United States
10.5
[22]
American Whites
United States
0.8
[19]
American Whites
United States
0
[22]
African Blacks
Uganda
>30
[23]
Arabs
Saudi Arabia
10.7
[24]
European Americans
United Kingdom
0.8
[19]
European Whites
United Kingdom
0
[22]
Yemenite Jews
Israel
11.8
[25]
Black Ethiopian Jews
Israel
15.4
[25]
Dominicans
Dominican Republic
0
[22]
Haitians
Haiti
8.2
[22]
Jamaicans
Jamaica
2.7
[22]
Text in bold indicates there was more than one study reported for the ethnic group.
Arabs, and Ethiopian and Yemenite Jews, in whom low neutrophil counts are common [19,22–25]. Benign neutropenia requires consideration in medical practice, in which white blood cell count is used as an indicator of immunocompetence, infection, and inflammation. This is particularly relevant in cases such as cancer clinical trials, in which participation based on white blood counts as one of the eligibility criteria into studies could exclude certain groups disproportionately. Although causes of benign neutropenia remain poorly understood, current consensus into the mechanisms behind neutropenia show no deficiency in the granulocyte colony-forming cells, normal myeloid maturation, and rather a slightly reduced number of bone marrow progenitor cells resulting in diminished physiologic release of mature neutrophils from vascular endothelium and the bone marrow stores to the periphery [13,21]. Recently, a genetic determinant for benign neutropenia was identified in African-Americans [26,27]. Using admixture mapping, two studies identified on chromosome 1q22 a polymorphism within the Duffy antigen receptor for chemokines (DARC) gene having a strong association with white blood cell counts in persons of African ancestry [26,27]. This polymorphism, whose allele frequency differs by 91% between Africans and Europeans, confers the DARC-negative (null) phenotype in which the DARC receptor is not expressed on erythrocytes. The DARC-null trait is associated with neutropenia and explains up to 20% of variation in white blood cells counts between African-Americans and European-Americans [27]. For example, He et al. [28] reported frequencies of 59% in male 52
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African-Americans, whereas 65% of men and women from South Africa possessed the DARC-null genotype [29]. The overall frequency of 47% has been reported in asymptomatic women of African origin in an American study: with percentages of 8, 63, 69, 73, and 75, respectively, in women of African descent from the Dominican Republic, Jamaica, Barbados, United States, and Haiti, respectively, whereas none of the white women of American or European origin had the DARC-null genotype [18]. In all studied women, the Duffy-null genotype was associated with lower absolute neutrophil counts. The DARC gene encodes for the Duffy antigen, a chemokine receptor found on red blood and endothelial cells. The receptor acts as ‘sink’ for proinflammatory chemokines, as these proteins can bind but not signal through this receptor. In this way, DARC may regulate chemokine levels in circulation and affect neutrophil localization, chemotaxis, and migration [13,30]. However, the mechanism of neutropenia in DARC-null trait individuals is unclear. In addition to the DARC gene polymorphism, additional genetic variants in CXCL2 on chromosome 4, near CDK6 on chromosome 7, and CSF3 on chromosome 17 are associated with low white blood cell counts in AfricanAmericans [31]. The rs9131 single-nucleotide polymorphism (SNP) located in the CXCL2 gene which encodes an inflammatory mediator involved in white blood cell production and migration is unique to African-Americans, whereas the remaining two SNPs in CDK6 and CSF3 are not unique to African-Americans [31]. The biological relevance and clinical implications of the DARC-null allele and several others were identified recently Volume 21 Number 1 January 2014
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Neutrophils in Africans Thobakgale and Ndung’u &&
in African ancestry [32 ]; the potential association of these genotypes with neutropenia and how they modulate progression to disease or susceptibility to infection warrants further investigation.
DUFFY-NULL GENOTYPE, NEUTROPENIA, AND DISEASE PATHOGENESIS Neutropenia occurs frequently during HIV-1 infection [33]. Interestingly, before the link between DARC-null genotype and neutropenia was demonstrated, it was shown that the DARC-null trait might play a role in HIV acquisition and disease progression [28]. It was reported that the DARC-null phenotype was associated with 40% increased likelihood of HIV acquisition, and it was postulated that considering the near-universal prevalence of the underlying DARC 46 C/C genotype in persons of African ancestry, it may account for up to 11% of the epidemic in sub-Saharan Africa [28]. Furthermore, this study demonstrated that following infection the DARC-null status was associated with slower disease progression. Whether these findings will hold true for African cohorts outside the USA remains an important question and could have implications on HIV pathogenesis, given the high prevalence of the DARC-null phenotype and low neutrophil counts in African ancestry. However, it is noteworthy that the role of the DARC-null trait on HIV pathogenesis is vigorously contested. Several cohort-based studies from diverse populations found no association with HIV-1 acquisition or progression to disease in AfricanAmericans and Africans, although differences in cohort designs and study end-points make direct comparisons between the studies difficult [29,34– 36]. We subsequently showed that low neutrophil counts before infection were associated with the DARC-null trait and an increased risk of HIV acquisition in black South African women [37]. Consistent with this finding, it has been reported that low neutrophil counts but not low lymphocytes or monocytes in mothers and children were associated with an increased risk of mother-to-childtransmission [38 ]. Thus, there is strong evidence emerging that neutropenia linked to DARC-null genotype plays a role in HIV transmission risk, although the mechanisms remain unclear. The impact of neutropenia and DARC-null phenotype on disease progression appears complicated and context dependent, with one study suggesting that neutropenia was associated with faster HIV disease progression in Europeans but with a survival advantage in African-Americans [39]. Therefore, the overall impact of the DARC-null trait and neutropenia on HIV pathogenesis remains unclear and is &&
likely to be affected by confounders. In this regard, a mechanistic approach to better understand the role of DARC in HIV pathogenesis may prove more informative than the genetic association studies because of the potential confounders with the latter. Several mechanisms have been proposed to explain how DARC may influence HIV pathogenesis. First, at the onset of infection, HIV-suppressive chemokines associated with DARC-expressing red blood cells may confer a protective shield on the receptor, preventing HIV attachment to erythrocytes and subsequent transfer to HIV target cells [28]. Alternatively, higher antiviral chemokines such as CCL5 in DARC-positive individuals may also be protective against the establishment of infection by a low-level inoculum. In contrast, following the establishment of infection, HIV may bind to DARC on red blood cells and be transported to cellular targets able to support productive infection, culminating in faster disease progression in DARCpositive persons. Furthermore, HIV thrives in an inflammatory milieu and DARC-positive individuals may be predisposed to a more pro-inflammatory state that promotes HIV replication and spread [39]. It is therefore speculated that slow disease progression in neutropenic individuals lacking DARC could in part be explained by reduced inflammatory state and binding of fewer HIV particles to DARC-null red blood cells [39]. Another example of infection outcome linked to DARC expression status relates to malaria infection. The DARC protein is used as a receptor for entry into red blood cells by the Plasmodium vivax malaria parasite, and the Duffy-null phenotype thus confers resistance to this parasite [40]. As a result, the protective DARC-null 46 C/C genotype has undergone positive selection in P. vivax malaria endemic areas, giving rise to its near-universal expression in Africa [41]. The role of neutrophils in malarial pathogenesis remains poorly understood, but there is accumulating evidence that neutrophil infiltration, activation, and dysfunction may worsen the infection outcomes or predispose to secondary bacterial infections [42–45]. These studies suggest that a major mechanism of poor outcome in malarial infection is neutrophil hyperactivation, and it is thus speculated that a similar mechanism that confers protection from neutrophil-mediated inflammation during malarial infection may operate during HIV infection, leading to the observed survival advantage in African persons with the DARC-null allele [39]. Although neutropenia is common in Africa, there are relatively few studies that have investigated the causes or consequences of this condition
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Myeloid biology Table 2. Variations associated with benign neutropenia in people of African descent and their impact on different diseases SNP
Location on chromosome
Candidate gene
Disease
Impact on disease and mechanism
rs2814778
1q23
DARC
HIV
Susceptibility to infection and slow disease progression
[28,39]
rs2814778
1q23
DARC
Plasmodium vivax
The absence of Duffy antigen results in lack of entry into red blood cells leading to resistance to infection by the malaria parasite
[40]
rs2814778
1q23
DARC
Plasmodium falciparum
Lack of platelet-mediated killing in the absence of Duffy antigen may enhance malaria disease
[46 ]
rs2814778
1q23
DARC
Sickle cell disease
Lack of Duffy antigen expression leads to organ damage and Duffy expression was associated with renal dysfunction
[47,48]
rs9131
4q13
CXCL2
Human obesity
High chemokine levels stimulate neutrophil adhesion and enhance white adipose tissue inflammation
[31]
rs445
7q21
CDK6
Rheumatoid arthritis
Associated with higher rate of joint destruction leading to susceptibility
[31]
rs4065321
17q21
PSMD3CFS3
Asthma
Associated with childhood onset of asthma disease
[31]
Several in both genes (see below)
1q41, 22q13.1
TGFB2, HMOX
Cerebral malaria
Protection from cerebral malaria disease mediated by neutrophilassociated, high-affinity receptor for IgE (IgE-FceRI)
[44]
rs1934852, rs6657275, rs1342586, rs1473527, rs4846478, rs6684205, rs1418553, rs900
1q41
TBFB2
Cerebral malaria
Protection from cerebral malaria disease mediated by neutrophilassociated, high-affinity receptor for IgE (IgE-FceRI)
[44]
rs2071748, rs8139532, rs7285877, rs11912889
22q13.1
HMOX
Cerebral malaria
Protection from cerebral malaria disease mediated by neutrophilassociated high-affinity receptor for IgE (IgE-FceRI)
[44]
References
&
SNP, single-nucleotide polymorphism.
(Table 2). An association of the DARC-null red blood cell phenotype as a potential biomarker of organ damage, particularly kidney dysfunction, has been reported during sickle cell disease [47,48]. In a recent study by McMorran et al. [46 ], the DARC receptor and platelet factor 4 (PF4/CXCL4) were identified to be crucial in the platelet-mediated killing of Plasmodium falciparum malaria parasites. This activity required DARC and was inhibited with the disruption of the DARC receptor expression. Therefore, unlike in the case of P. vivax, where the DARC-null genotype is protective against infection, the same genotype may be detrimental in P. falciparum infection because of the lack of platelet-mediated parasite killing. Collectively, these observations call for new studies to understand the role of the DARC-null genotype and low neutrophil counts on various diseases as illustrated in Fig. 1. Such new findings could identify potential biomarkers of disease protection or severity, and
could also motivate identification of race-specific reference ranges and therapies for people of African descent to prevent poor outcomes of different illnesses.
&
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NEUTROPHILS AGAINST INTRACELLULAR PATHOGENS Although neutrophils are immune mediators against bacterial and fungal pathogens, their role against viruses and other intracellular pathogens is only beginning to emerge. In tuberculosis (TB) infection, neutrophils have been suggested to downregulate inflammation during the early stages of infection while promoting inflammation during chronic stages [49]. Engulfment of dying cells at the site of infection by neutrophils during granuloma formation was found to contribute to the containment of mycobacterial infection [50 ]. Neutrophils are thought to contribute to innate resistance to &
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Neutrophils in Africans Thobakgale and Ndung’u
(a)
DARC-null trait
Other genetic and environmental factors
(b)
Other genetic and environmental factors
Favourable cytokine/ chemokine balance? Other mechanisms?
Mechanisms?
Increased pro-inflammatory chemokine levels? Other mechanisms?
DARC-positive
Mechanisms?
T cell
T cell
B cell Reduced pathogen killing via NETosis, phagocytosis and degranulation
B cell Optimal pathogen killing via NETosis, phagocytosis and degranulation
Reduced priming and immune interactions NK cell
Optimal priming and immune interactions NK cell
Platelet
Platelet
Macrophage/ dendritic cell
Macrophage/ dendritic cell
Increased susceptibility or failure to control HIV, malaria ( ), TB and other diseases?
Less infections and better control of HIV, malaria ( ), TB and other diseases?
FIGURE 1. A simplified model of how neutrophil counts in Africans may affect susceptibility or ability to control diseases. (a) In the context of neutropenia, caused by genetic or environmental factors, neutrophils are unable to clear infection and there may be inability to prime innate and adaptive immune cells, leading to increased risk of infection and pathogen spread. (b) When neutrophils counts and functions are optimal as dictated by genetic and other undetermined factors, there is enhanced pathogen clearance, and neutrophil priming and cross-talk with other immune cells result in desired clinical outcome. It should be noted that this schema represents a simplified model as neutrophil infiltration and burst can exacerbate disease leading hypothetically to better outcomes under neutropenia for certain diseases. Experimental data are required to critically test and refine this model.
TB infection and may also actively fight against mycobacteria [51]. The role of neutrophils in the host defense against viral pathogens has not been well studied. A few recent studies have suggested a role for NETs in the control of HIV-1 [52 ,53 ]. Priming of virusspecific T cells by neutrophils has been suggested in another study [54 ]. Thus, neutrophils may have a protective or harmful role, and better understanding of their wider role in infectious diseases, as illustrated in Fig. 1, may provide new avenues for immunotherapeutic intervention. &
&
&
CONCLUSION Benign neutropenia is very common in people of African descent and the consequence of this variation from normal counts is not known. Several reports in the recent past have suggested a link between DARC genotype, neutrophils, and various diseases common in Africa, highlighting the need to understand the possible interactions and mechanisms. Studies on the role of neutrophils in disease pathogenesis are likely to have significant public
health implications on populations of sub-Saharan Africa, in which there is not only high infectious disease burden, including HIV and AIDS, TB, and malaria, but also near-universal expression of the DARC-null genotype associated with low neutrophil counts. The mechanisms underlying the interaction between the DARC genotype and white blood cell counts, and how this may influence the function and cross-talk of neutrophils with other immune cells such as T cells, NK cells, and macrophages will form an important part of future research as these new studies provide new avenues in understanding neutrophils and how they may respond to pathogens. Acknowledgements The authors would like to thank Kewreshini Naidoo for assistance with literature scans and Siyabonga Nikwe for assistance with graphic illustrations. Research work in the authors’ laboratory is funded by the South African Research Chairs Initiative of the South African Department of Science and Technology through the National Research Foundation. Additional funding was received from the International AIDS Vaccine
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Myeloid biology
Initiative, the Victor Daitz Chair in HIV/TB Research, and an International Early Career Scientist Award from the Howard Hughes Medical Institute. Conflicts of interest There are no conflicts of interest.
REFERENCES AND RECOMMENDED READING Papers of particular interest, published within the annual period of review, have been highlighted as: & of special interest && of outstanding interest 1. Mestas J, Hughes CC. Of mice and not men: differences between mouse and human immunology. J Immunol 2004; 172:2731–2738. 2. Borregaard N. Neutrophils, from marrow to microbes. Immunity 2010; 33:657–670. 3. Bugl S, Wirths S, Muller MR, et al. Current insights into neutrophil homeostasis. Ann N Y Acad Sci 2012; 1266:171–178. 4. Summers C, Rankin SM, Condliffe AM, et al. Neutrophil kinetics in health and disease. Trends Immunol 2010; 31:318–324. 5. Amulic B, Cazalet C, Hayes GL, et al. Neutrophil function: from mechanisms to && disease. Annu Rev Immunol 2012; 30:459–489. An excellet review summarizing the new functions of neutrophils beyond inflammation as regulators of the immune system. 6. Mocsai A. Diverse novel functions of neutrophils in immunity, inflammation, && and beyond. J Exp Med 2013; 210:1283–1299. Another excellent recent article highlighting the novel and sophisticated functions of neutrophil functions beyond inflammation. 7. Boudaly S. Activation of dendritic cells by polymorphonuclear neutrophils. Front Biosci 2009; 14:1589–1595. 8. Silva MT. Neutrophils and macrophages work in concert as inducers and effectors of adaptive immunity against extracellular and intracellular microbial pathogens. J Leukoc Biol 2010; 87:805–813. 9. Costantini C, Cassatella MA. The defensive alliance between neutrophils and NK cells as a novel arm of innate immunity. J Leukoc Biol 2011; 89:221– 233. 10. Scapini P, Bazzoni F, Cassatella MA. Regulation of B-cell-activating factor (BAFF)/B lymphocyte stimulator (BLyS) expression in human neutrophils. Immunol Lett 2008; 116:1–6. 11. Muller I, Munder M, Kropf P, et al. Polymorphonuclear neutrophils and T lymphocytes: strange bedfellows or brothers in arms? Trends Immunol 2009; 30:522–530. 12. Haddy TB, Rana SR, Castro O. Benign ethnic neutropenia: what is a normal absolute neutrophil count? J Lab Clin Med 1999; 133:15–22. 13. Paz Z, Nails M, Ziv E. The genetics of benign neutropenia. Isr Med Assoc J 2011; 13:625–629. 14. Donadieu J, Fenneteau O, Beaupain B, et al. Congenital neutropenia: diagnosis, molecular bases and patient management. Orphanet J Rare Dis 2011; 6:26. 15. Boxer LA. How to approach neutropenia? Hematology Am Soc Hematol Educ && Program 2012; 2012:174–182. This article enhances the general understanding of different types of neutropenia, cause, and implication in clinical settings. 16. Rezaei N, Moazzami K, Aghamohammadi A, et al. Neutropenia and primary immunodeficiency diseases. Int Rev Immunol 2009; 28:335–366. 17. Sokolic R. Neutropenia in primary immunodeficiency. Curr Opin Hematol & 2013; 20:55–65. The genetic defects associated with congenital neutropenia-associated disorders are summarized in this review. 18. Grann VR, Ziv E, Joseph CK, et al. Duffy (Fy), DARC, and neutropenia among women from the United States, Europe and the Caribbean. Br J Haematol 2008; 143:288–293. 19. Hsieh MM, Everhart JE, Byrd-Holt DD, et al. Prevalence of neutropenia in the U.S. population: age, sex, smoking status, and ethnic differences. Ann Intern Med 2007; 146:486–492. 20. Shoenfeld Y, Ben-Tal O, Berliner S, et al. The outcome of bacterial infection in subjects with benign familial leukopenia (BFL). Biomed Pharmacother 1985; 39:23–26. 21. Hsieh MM, Tisdale JF, Rodgers GP, et al. Neutrophil count in African Americans: lowering the target cutoff to initiate or resume chemotherapy? J Clin Oncol 2010; 28:1633–1637. 22. Grann VR, Bowman N, Joseph C, et al. Neutropenia in 6 ethnic groups from the Caribbean and the U.S. Cancer 2008; 113:854–860. 23. Shaper AG, Lewis P. Genetic neutropenia in people of African origin. Lancet 1971; 2:1021–1023.
56
www.co-hematology.com
24. Denic S, Showqi S, Klein C, et al. Prevalence, phenotype and inheritance of benign neutropenia in Arabs. BMC Blood Disord 2009; 9:3. 25. Weingarten MA, Pottick-Schwartz EA, Brauner A. The epidemiology of benign leukopenia in Yemenite Jews. Isr J Med Sci 1993; 29:297– 299. 26. Nalls MA, Wilson JG, Patterson NJ, et al. Admixture mapping of white cell count: genetic locus responsible for lower white blood cell count in the Health ABC and Jackson Heart studies. Am J Hum Genet 2008; 82:81–87. 27. Reich D, Nalls MA, Kao WH, et al. Reduced neutrophil count in people of African descent is due to a regulatory variant in the Duffy antigen receptor for chemokines gene. PLoS Genet 2009; 5:e1000360. 28. He W, Neil S, Kulkarni H, et al. Duffy antigen receptor for chemokines mediates trans-infection of HIV-1 from red blood cells to target cells and affects HIV-AIDS susceptibility. Cell Host Microbe 2008; 4:52–62. 29. Julg B, Reddy S, van der Stok M, et al. Lack of Duffy antigen receptor for chemokines: no influence on HIV disease progression in an African treatmentnaive population. Cell Host Microbe 2009; 5:413–415; author reply 8–9. 30. Lee JS, Frevert CW, Wurfel MM, et al. Duffy antigen facilitates movement of chemokine across the endothelium in vitro and promotes neutrophil transmigration in vitro and in vivo. J Immunol 2003; 170:5244–5251. 31. Reiner AP, Lettre G, Nalls MA, et al. Genome-wide association study of white blood cell count in 16 388 African Americans: the Continental Origins and Genetic Epidemiology Network (COGENT). PLoS Genet 2011; 7:e1002108. 32. Crosslin DR, McDavid A, Weston N, et al. Genetic variants associated && with the white blood cell count in 13 923 subjects in the eMERGE Network. Hum Genet 2012; 131:639–652. This study also conducted in large cohorts of European and African Americans confirmed the association of the DARC-null trait to white blood cell counts. In addition, the study identified for the first time that other SNPs previously associated with asthma were also associated with white blood cell counts. 33. Levine AM, Karim R, Mack W, et al. Neutropenia in human immunodeficiency virus infection: data from the women’s interagency HIV study. Arch Intern Med 2006; 166:405–410. 34. Horne KC, Li X, Jacobson LP, et al. Duffy antigen polymorphisms do not alter progression of HIV in African Americans in the MACS cohort. Cell Host Microbe 2009; 5:415–417; author reply 8–9. 35. Walley NM, Julg B, Dickson SP, et al. The Duffy antigen receptor for chemokines null promoter variant does not influence HIV-1 acquisition or disease progression. Cell Host Microbe 2009; 5:408–410; author reply 18–19. 36. Winkler CA, An P, Johnson R, et al. Expression of Duffy antigen receptor for chemokines (DARC) has no effect on HIV-1 acquisition or progression to AIDS in African Americans. Cell Host Microbe 2009; 5:411–413; author reply 8–9. 37. Ramsuran V, Kulkarni H, He W, et al. Duffy-null-associated low neutrophil counts influence HIV-1 susceptibility in high-risk South African black women. Clin Infect Dis 2011; 52:1248–1256. 38. Kourtis AP, Hudgens MG, Kayira D. Neutrophil count in African mothers && and newborns and HIV transmission risk. N Engl J Med 2012; 367:2260– 2262. Higher neutrophil counts in the mother and in newborn were found to be associated with lower risk of mother-to-child HIV transmission in a study conducted in Malawi. 39. Kulkarni H, Marconi VC, He W, et al. The Duffy-null state is associated with a survival advantage in leukopenic HIV-infected persons of African ancestry. Blood 2009; 114:2783–2792. 40. Horuk R, Chitnis CE, Darbonne WC, et al. A receptor for the malarial parasite Plasmodium vivax: the erythrocyte chemokine receptor. Science 1993; 261:1182–1184. 41. Tournamille C, Blancher A, Le Van Kim C, et al. Sequence, evolution and ligand binding properties of mammalian Duffy antigen/receptor for chemokines. Immunogenetics 2004; 55:682–694. 42. Cunnington AJ, Njie M, Correa S, et al. Prolonged neutrophil dysfunction after Plasmodium falciparum malaria is related to hemolysis and heme oxygenase-1 induction. J Immunol 2012; 189:5336–5346. 43. Dey S, Bindu S, Goyal M, et al. Impact of intravascular hemolysis in malaria on liver dysfunction: involvement of hepatic free heme overload, NF-kappaB activation, and neutrophil infiltration. J Biol Chem 2012; 287:26630– 26646. 44. Porcherie A, Mathieu C, Peronet R, et al. Critical role of the neutrophilassociated high-affinity receptor for IgE in the pathogenesis of experimental cerebral malaria. J Exp Med 2011; 208:2225–2236. 45. Walther M, De Caul A, Aka P, et al. HMOX1 gene promoter alleles and high HO-1 levels are associated with severe malaria in Gambian children. PLoS Pathog 2012; 8:e1002579. 46. McMorran BJ, Wieczorski L, Drysdale KE, et al. Platelet factor 4 and Duffy & antigen required for platelet killing of Plasmodium falciparum. Science 2012; 338:1348–1351. DARC receptor essential for binding of platelet factor 4 (PF4) leading to efficient platelet-mediated killing of Plasmodium falciparum. 47. Afenyi-Annan A, Kail M, Combs MR, et al. Lack of Duffy antigen expression is associated with organ damage in patients with sickle cell disease. Transfusion 2008; 48:917–924.
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Neutrophils in Africans Thobakgale and Ndung’u 48. Drasar ER, Menzel S, Fulford T, et al. The effect of Duffy antigen receptor for chemokines on severity in sickle cell disease. Haematologica 2013; 98:e87–e89. 49. Zhang X, Majlessi L, Deriaud E, et al. Coactivation of Syk kinase and MyD88 adaptor protein pathways by bacteria promotes regulatory properties of neutrophils. Immunity 2009; 31:761–771. 50. Yang CT, Cambier CJ, Davis JM, et al. Neutrophils exert protection in the early tuberculous granuloma by oxidative killing of mycobacteria phagocytosed from infected macrophages. Cell Host Microbe 2012; 12:301– 312. Neutrophils contribute to containing mycobacteria infection through engulfment of dying cells within the granuloma rather than by interaction with the microbes.
&
51. Martineau AR, Newton SM, Wilkinson KA, et al. Neutrophil-mediated innate immune resistance to mycobacteria. J Clin Invest 2007; 117:1988– 1994.
52. Jenne CN, Wong CH, Zemp FJ, et al. Neutrophils recruited to sites of infection protect from virus challenge by releasing neutrophil extracellular traps. Cell Host Microbe 2013; 13:169–180. A second study to suggest a protective role of neutrophils in innate antiviral immunity. Poxvirus infection led to the recruitment of neutrophils and platelets to the liver sinusoids, forming NETs and in the process protecting host cells from infection. 53. Saitoh T, Komano J, Saitoh Y, et al. Neutrophil extracellular traps mediate & a host defense response to human immunodeficiency virus-1. Cell Host Microbe 2012; 12:109–116. One of the first studies to demonstrate the ability of neutrophils to eliminate HIV and mediate an antiviral response through the formation of NETs. 54. Duffy D, Perrin H, Abadie V, et al. Neutrophils transport antigen from the & dermis to the bone marrow, initiating a source of memory CD8þ T cells. Immunity 2012; 37:917–929. A role of neutrophils in the antigen-specific T-cell response is suggested in mouse models, in which neutrophils are shown to be virus carrier cells from the dermis to the bone marrow initiating a source of memory CD8þ T cells. &
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Neutrophil counts in persons of African origin Christina F. Thobakgale a,b,c and Thumbi Ndung’u a,b,c,d
Purpose of review The causes of ethnic or benign neutropenia have long been unclear. Here, we discuss the emerging data on the causes and consequences of neutropenia and discuss the relevance of these data for African populations, in which the prevalence of neutropenia is high. Recent findings Genetic deletion of the Duffy antigen receptor for chemokines (DARC-null genotype) has been identified as a major determinant for neutropenia. DARC acts as a receptor for Plasmodium vivax malaria and the DARC-null genotype has thus been positively selected among Africans; however, recent studies suggest that Duffy-null-linked neutropenia could increase the risk of HIV infection. Data are emerging that neutrophils are versatile cells that play a critical role not only in direct antimicrobial activity but also in priming and regulating the activity of other innate and adaptive immune cells. Therefore, we discuss here the imperative to better understand the causes, consequences, and the underlying mechanisms of neutropenia among Africans as a prerequisite for rational and optimal biomedical interventions to improve health outcomes. Summary Neutropenia among Africans, linked to the Duffy-null trait or otherwise, may have significant health consequences that remain largely undetermined and could have a significant impact on the pathogenesis of diseases. Keywords Africans, antimicrobial immunity, Duffy-null, neutrophils, phenotype
INTRODUCTION Neutrophils have emerged as versatile cells capable of direct antimicrobial activity and immune regulation. Thus, a reduction in their numbers as seen in rare cases of severe congenital neutropenia can have life-threatening implications. In contrast, ethnic or benign neutropenia is common among Africans and is thought to be of no adverse clinical impact. In this review, we address the recent findings demonstrating that neutrophils play a critical role in controlling infection and inflammation. We discuss data on the genetic basis of congenital and benign neutropenia and association of neutropenia with infection and disease outcomes. We also highlight that there is paucity of data on the causes and implication of benign neutropenia on disease and propose that epidemiological, genetic, and mechanistic studies will be required to unravel whether this condition is detrimental or beneficial in specific circumstances. This research is needed to optimize interventions and improve health outcomes in African ancestry populations. www.co-hematology.com
NEUTROPHILS ON THE FRONT LINE AND AS REGULATORS OF ADAPTIVE IMMUNITY Neutrophils, also known as polymorphonuclear leukocytes (PMNs), are innate immune cells, approximately 7–10 mm in diameter, and characterized morphologically by the presence of a segmented nucleus and cytoplasmic granules and secretory a
HIV Pathogenesis Programme, Doris Duke Medical Research Institute, KwaZulu-Natal Research Institute for Tuberculosis and HIV, Nelson R. Mandela School of Medicine, University of KwaZulu-Natal, Durban, South Africa, cRagon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology, and Harvard University, Boston, Massachusetts, USA and dMax Planck Institute for Infection Biology, Berlin, Germany b
Correspondence to Thumbi Ndung’u, PhD, HIV Pathogenesis Programme, Doris Duke Medical Research Institute, Nelson R. Mandela School of Medicine, University of KwaZulu-Natal, 719 Umbilo Road, Durban, 4013, South Africa. Tel: +27 31 260 4727; fax: +27 31 260 4623; e-mail: [email protected] Curr Opin Hematol 2014, 21:50–57 DOI:10.1097/MOH.0000000000000007 Volume 21 Number 1 January 2014
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Neutrophils in Africans Thobakgale and Ndung’u
KEY POINTS Neutrophils can inhibit various pathogens directly through phagocytosis, degranulation, and NETosis. Neutrophils also interact with other immune cells such as T cells, B cells, macrophages, natural killer cells, platelets, and dendritic cells. These interactions can shape the subsequent immune outcomes. Benign neutropenia is common among Africans and considering the important role that neutrophils play in disease pathogenesis, this condition could be detrimental or beneficial depending on the particular pathogen and host characteristics. The biological basis and consequences of ethnic neutropenia require further research for improved biomedical interventions.
vesicles. Neutrophils originate from precursor myeloid cells in the bone marrow, where they differentiate into myeloblasts before maturing into neutrophils. With about 1–2 1011 cells produced per day, they are the most abundant white blood cells in humans and constitute about 50–70% of leukocytes in circulation [1], and are classically known for the role they play in acute immune response against bacterial and fungal infections. The chemokine receptor CXCR4 (ligand SDF-1) is crucial for maintaining neutrophils in the bone marrow, whereas CXCR2 (ligands KC and Grob) is responsible for neutrophil release. Granulocyte colony-stimulating factor (G-CSF) stimulates neutrophil release directly and indirectly by reducing SDF-1 expression and enhancing the expression of Grob on endothelial cells [2]. Thus, neutrophil homeostasis is maintained by a fine balance between production, storage, and release of neutrophils from the bone marrow, margination, and clearance [3,4]. Neutrophils engulf and eliminate their pathogens by the mechanisms of phagocytosis, degranulation, or formation of neutrophil extracellular traps (NETs) [5 ,6 ]. Emerging reports over the last few years indicate that neutrophils have immune functions beyond phagocytosis and resolving inflammation. In addition to producing cytokines and chemokines [5 ], neutrophils also engage in bidirectional interactions with other immune cells and are emerging as regulators of the innate and adaptive immune responses. The neutrophil’s interaction with other immune cells, including macrophages [7], dendritic cells [8], natural killer (NK) cells [9], B cells [10], and T lymphocytes [11], has only come to light in recent studies and is still poorly understood; however, &&
&&
&&
these studies highlight the potential importance of neutrophils in regulating innate and adaptive immune responses.
NEUTROPHILS IN DISEASE Given the abundance and important role of neutrophils as the first line of immune defense against pathogens and as regulators of innate and adaptive immunity, it is not surprising that total absence of neutrophils or marked decrease in their numbers results in profound immunodeficiencies. Neutropenia is defined as a reduction in absolute neutrophil counts to below 1500 cells/ml for older children and adults regardless of ethnicity [12]. Neutropenia can be stratified according to the clinical severity with absolute neutrophil counts between 1000 and 1500 cells/ml being defined as mild neutropenia, 500–1000 cells/ml is considered moderate neutropenia, whereas counts below 500 cells/ml is termed severe neutropenia [13]. The risk of neutropeniaassociated disorders greatly increases with absolute neutrophil counts below 500 cells/ml. Acute neutropenia, defined as normally resolvable short-term drop in absolute neutrophil counts because of infection or injury, occurs very frequently compared with congenital or cyclic neutropenia which occur only rarely in the general population at 6.2 cases per million [14]. The biological basis, clinical presentation, and management of congenital and cyclic neutropenic disorders have been discussed in detail by others [15 ,16,17 ]. &&
&
FEATURES AND GENETICS OF ETHNIC BENIGN NEUTROPENIA Neutrophil counts vary greatly by ethnic groups. Low absolute counts are a common occurrence in people of African descent and some ethnic groups from the Middle East, a condition known as ethnic or benign neutropenia. Benign ethnic neutropenia occurs in 25–50% persons of African descent and does not appear to be associated with clinical disadvantage compared with matched population controls [12,18–20]. The condition is diagnosed by repeated low measurements of absolute neutrophil counts less than 1500 cells/ml (usually 1000 cells/ml or below) for several months without identifiable causes of neutropenia. Unlike congenital neutropenia, usually diagnosed among whites and potentially life threatening, ethnic neutropenia is clinically inconsequential as it is not associated with increased risk of mucosal or systemic infections [21]. The reported prevalence of benign neutropenia in different ethnic groups is summarized in Table 1 and includes reports from peoples of African descent,
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Myeloid biology Table 1. Prevalence of benign neutropenia by ethnic populations Ethnic group
Country of origin
Benign neutropenia prevalence (%)
Reference
American Blacks
United States
4.5
[19]
American Blacks
United States
10.5
[22]
American Whites
United States
0.8
[19]
American Whites
United States
0
[22]
African Blacks
Uganda
>30
[23]
Arabs
Saudi Arabia
10.7
[24]
European Americans
United Kingdom
0.8
[19]
European Whites
United Kingdom
0
[22]
Yemenite Jews
Israel
11.8
[25]
Black Ethiopian Jews
Israel
15.4
[25]
Dominicans
Dominican Republic
0
[22]
Haitians
Haiti
8.2
[22]
Jamaicans
Jamaica
2.7
[22]
Text in bold indicates there was more than one study reported for the ethnic group.
Arabs, and Ethiopian and Yemenite Jews, in whom low neutrophil counts are common [19,22–25]. Benign neutropenia requires consideration in medical practice, in which white blood cell count is used as an indicator of immunocompetence, infection, and inflammation. This is particularly relevant in cases such as cancer clinical trials, in which participation based on white blood counts as one of the eligibility criteria into studies could exclude certain groups disproportionately. Although causes of benign neutropenia remain poorly understood, current consensus into the mechanisms behind neutropenia show no deficiency in the granulocyte colony-forming cells, normal myeloid maturation, and rather a slightly reduced number of bone marrow progenitor cells resulting in diminished physiologic release of mature neutrophils from vascular endothelium and the bone marrow stores to the periphery [13,21]. Recently, a genetic determinant for benign neutropenia was identified in African-Americans [26,27]. Using admixture mapping, two studies identified on chromosome 1q22 a polymorphism within the Duffy antigen receptor for chemokines (DARC) gene having a strong association with white blood cell counts in persons of African ancestry [26,27]. This polymorphism, whose allele frequency differs by 91% between Africans and Europeans, confers the DARC-negative (null) phenotype in which the DARC receptor is not expressed on erythrocytes. The DARC-null trait is associated with neutropenia and explains up to 20% of variation in white blood cells counts between African-Americans and European-Americans [27]. For example, He et al. [28] reported frequencies of 59% in male 52
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African-Americans, whereas 65% of men and women from South Africa possessed the DARC-null genotype [29]. The overall frequency of 47% has been reported in asymptomatic women of African origin in an American study: with percentages of 8, 63, 69, 73, and 75, respectively, in women of African descent from the Dominican Republic, Jamaica, Barbados, United States, and Haiti, respectively, whereas none of the white women of American or European origin had the DARC-null genotype [18]. In all studied women, the Duffy-null genotype was associated with lower absolute neutrophil counts. The DARC gene encodes for the Duffy antigen, a chemokine receptor found on red blood and endothelial cells. The receptor acts as ‘sink’ for proinflammatory chemokines, as these proteins can bind but not signal through this receptor. In this way, DARC may regulate chemokine levels in circulation and affect neutrophil localization, chemotaxis, and migration [13,30]. However, the mechanism of neutropenia in DARC-null trait individuals is unclear. In addition to the DARC gene polymorphism, additional genetic variants in CXCL2 on chromosome 4, near CDK6 on chromosome 7, and CSF3 on chromosome 17 are associated with low white blood cell counts in AfricanAmericans [31]. The rs9131 single-nucleotide polymorphism (SNP) located in the CXCL2 gene which encodes an inflammatory mediator involved in white blood cell production and migration is unique to African-Americans, whereas the remaining two SNPs in CDK6 and CSF3 are not unique to African-Americans [31]. The biological relevance and clinical implications of the DARC-null allele and several others were identified recently Volume 21 Number 1 January 2014
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Neutrophils in Africans Thobakgale and Ndung’u &&
in African ancestry [32 ]; the potential association of these genotypes with neutropenia and how they modulate progression to disease or susceptibility to infection warrants further investigation.
DUFFY-NULL GENOTYPE, NEUTROPENIA, AND DISEASE PATHOGENESIS Neutropenia occurs frequently during HIV-1 infection [33]. Interestingly, before the link between DARC-null genotype and neutropenia was demonstrated, it was shown that the DARC-null trait might play a role in HIV acquisition and disease progression [28]. It was reported that the DARC-null phenotype was associated with 40% increased likelihood of HIV acquisition, and it was postulated that considering the near-universal prevalence of the underlying DARC 46 C/C genotype in persons of African ancestry, it may account for up to 11% of the epidemic in sub-Saharan Africa [28]. Furthermore, this study demonstrated that following infection the DARC-null status was associated with slower disease progression. Whether these findings will hold true for African cohorts outside the USA remains an important question and could have implications on HIV pathogenesis, given the high prevalence of the DARC-null phenotype and low neutrophil counts in African ancestry. However, it is noteworthy that the role of the DARC-null trait on HIV pathogenesis is vigorously contested. Several cohort-based studies from diverse populations found no association with HIV-1 acquisition or progression to disease in AfricanAmericans and Africans, although differences in cohort designs and study end-points make direct comparisons between the studies difficult [29,34– 36]. We subsequently showed that low neutrophil counts before infection were associated with the DARC-null trait and an increased risk of HIV acquisition in black South African women [37]. Consistent with this finding, it has been reported that low neutrophil counts but not low lymphocytes or monocytes in mothers and children were associated with an increased risk of mother-to-childtransmission [38 ]. Thus, there is strong evidence emerging that neutropenia linked to DARC-null genotype plays a role in HIV transmission risk, although the mechanisms remain unclear. The impact of neutropenia and DARC-null phenotype on disease progression appears complicated and context dependent, with one study suggesting that neutropenia was associated with faster HIV disease progression in Europeans but with a survival advantage in African-Americans [39]. Therefore, the overall impact of the DARC-null trait and neutropenia on HIV pathogenesis remains unclear and is &&
likely to be affected by confounders. In this regard, a mechanistic approach to better understand the role of DARC in HIV pathogenesis may prove more informative than the genetic association studies because of the potential confounders with the latter. Several mechanisms have been proposed to explain how DARC may influence HIV pathogenesis. First, at the onset of infection, HIV-suppressive chemokines associated with DARC-expressing red blood cells may confer a protective shield on the receptor, preventing HIV attachment to erythrocytes and subsequent transfer to HIV target cells [28]. Alternatively, higher antiviral chemokines such as CCL5 in DARC-positive individuals may also be protective against the establishment of infection by a low-level inoculum. In contrast, following the establishment of infection, HIV may bind to DARC on red blood cells and be transported to cellular targets able to support productive infection, culminating in faster disease progression in DARCpositive persons. Furthermore, HIV thrives in an inflammatory milieu and DARC-positive individuals may be predisposed to a more pro-inflammatory state that promotes HIV replication and spread [39]. It is therefore speculated that slow disease progression in neutropenic individuals lacking DARC could in part be explained by reduced inflammatory state and binding of fewer HIV particles to DARC-null red blood cells [39]. Another example of infection outcome linked to DARC expression status relates to malaria infection. The DARC protein is used as a receptor for entry into red blood cells by the Plasmodium vivax malaria parasite, and the Duffy-null phenotype thus confers resistance to this parasite [40]. As a result, the protective DARC-null 46 C/C genotype has undergone positive selection in P. vivax malaria endemic areas, giving rise to its near-universal expression in Africa [41]. The role of neutrophils in malarial pathogenesis remains poorly understood, but there is accumulating evidence that neutrophil infiltration, activation, and dysfunction may worsen the infection outcomes or predispose to secondary bacterial infections [42–45]. These studies suggest that a major mechanism of poor outcome in malarial infection is neutrophil hyperactivation, and it is thus speculated that a similar mechanism that confers protection from neutrophil-mediated inflammation during malarial infection may operate during HIV infection, leading to the observed survival advantage in African persons with the DARC-null allele [39]. Although neutropenia is common in Africa, there are relatively few studies that have investigated the causes or consequences of this condition
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Myeloid biology Table 2. Variations associated with benign neutropenia in people of African descent and their impact on different diseases SNP
Location on chromosome
Candidate gene
Disease
Impact on disease and mechanism
rs2814778
1q23
DARC
HIV
Susceptibility to infection and slow disease progression
[28,39]
rs2814778
1q23
DARC
Plasmodium vivax
The absence of Duffy antigen results in lack of entry into red blood cells leading to resistance to infection by the malaria parasite
[40]
rs2814778
1q23
DARC
Plasmodium falciparum
Lack of platelet-mediated killing in the absence of Duffy antigen may enhance malaria disease
[46 ]
rs2814778
1q23
DARC
Sickle cell disease
Lack of Duffy antigen expression leads to organ damage and Duffy expression was associated with renal dysfunction
[47,48]
rs9131
4q13
CXCL2
Human obesity
High chemokine levels stimulate neutrophil adhesion and enhance white adipose tissue inflammation
[31]
rs445
7q21
CDK6
Rheumatoid arthritis
Associated with higher rate of joint destruction leading to susceptibility
[31]
rs4065321
17q21
PSMD3CFS3
Asthma
Associated with childhood onset of asthma disease
[31]
Several in both genes (see below)
1q41, 22q13.1
TGFB2, HMOX
Cerebral malaria
Protection from cerebral malaria disease mediated by neutrophilassociated, high-affinity receptor for IgE (IgE-FceRI)
[44]
rs1934852, rs6657275, rs1342586, rs1473527, rs4846478, rs6684205, rs1418553, rs900
1q41
TBFB2
Cerebral malaria
Protection from cerebral malaria disease mediated by neutrophilassociated, high-affinity receptor for IgE (IgE-FceRI)
[44]
rs2071748, rs8139532, rs7285877, rs11912889
22q13.1
HMOX
Cerebral malaria
Protection from cerebral malaria disease mediated by neutrophilassociated high-affinity receptor for IgE (IgE-FceRI)
[44]
References
&
SNP, single-nucleotide polymorphism.
(Table 2). An association of the DARC-null red blood cell phenotype as a potential biomarker of organ damage, particularly kidney dysfunction, has been reported during sickle cell disease [47,48]. In a recent study by McMorran et al. [46 ], the DARC receptor and platelet factor 4 (PF4/CXCL4) were identified to be crucial in the platelet-mediated killing of Plasmodium falciparum malaria parasites. This activity required DARC and was inhibited with the disruption of the DARC receptor expression. Therefore, unlike in the case of P. vivax, where the DARC-null genotype is protective against infection, the same genotype may be detrimental in P. falciparum infection because of the lack of platelet-mediated parasite killing. Collectively, these observations call for new studies to understand the role of the DARC-null genotype and low neutrophil counts on various diseases as illustrated in Fig. 1. Such new findings could identify potential biomarkers of disease protection or severity, and
could also motivate identification of race-specific reference ranges and therapies for people of African descent to prevent poor outcomes of different illnesses.
&
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NEUTROPHILS AGAINST INTRACELLULAR PATHOGENS Although neutrophils are immune mediators against bacterial and fungal pathogens, their role against viruses and other intracellular pathogens is only beginning to emerge. In tuberculosis (TB) infection, neutrophils have been suggested to downregulate inflammation during the early stages of infection while promoting inflammation during chronic stages [49]. Engulfment of dying cells at the site of infection by neutrophils during granuloma formation was found to contribute to the containment of mycobacterial infection [50 ]. Neutrophils are thought to contribute to innate resistance to &
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Neutrophils in Africans Thobakgale and Ndung’u
(a)
DARC-null trait
Other genetic and environmental factors
(b)
Other genetic and environmental factors
Favourable cytokine/ chemokine balance? Other mechanisms?
Mechanisms?
Increased pro-inflammatory chemokine levels? Other mechanisms?
DARC-positive
Mechanisms?
T cell
T cell
B cell Reduced pathogen killing via NETosis, phagocytosis and degranulation
B cell Optimal pathogen killing via NETosis, phagocytosis and degranulation
Reduced priming and immune interactions NK cell
Optimal priming and immune interactions NK cell
Platelet
Platelet
Macrophage/ dendritic cell
Macrophage/ dendritic cell
Increased susceptibility or failure to control HIV, malaria ( ), TB and other diseases?
Less infections and better control of HIV, malaria ( ), TB and other diseases?
FIGURE 1. A simplified model of how neutrophil counts in Africans may affect susceptibility or ability to control diseases. (a) In the context of neutropenia, caused by genetic or environmental factors, neutrophils are unable to clear infection and there may be inability to prime innate and adaptive immune cells, leading to increased risk of infection and pathogen spread. (b) When neutrophils counts and functions are optimal as dictated by genetic and other undetermined factors, there is enhanced pathogen clearance, and neutrophil priming and cross-talk with other immune cells result in desired clinical outcome. It should be noted that this schema represents a simplified model as neutrophil infiltration and burst can exacerbate disease leading hypothetically to better outcomes under neutropenia for certain diseases. Experimental data are required to critically test and refine this model.
TB infection and may also actively fight against mycobacteria [51]. The role of neutrophils in the host defense against viral pathogens has not been well studied. A few recent studies have suggested a role for NETs in the control of HIV-1 [52 ,53 ]. Priming of virusspecific T cells by neutrophils has been suggested in another study [54 ]. Thus, neutrophils may have a protective or harmful role, and better understanding of their wider role in infectious diseases, as illustrated in Fig. 1, may provide new avenues for immunotherapeutic intervention. &
&
&
CONCLUSION Benign neutropenia is very common in people of African descent and the consequence of this variation from normal counts is not known. Several reports in the recent past have suggested a link between DARC genotype, neutrophils, and various diseases common in Africa, highlighting the need to understand the possible interactions and mechanisms. Studies on the role of neutrophils in disease pathogenesis are likely to have significant public
health implications on populations of sub-Saharan Africa, in which there is not only high infectious disease burden, including HIV and AIDS, TB, and malaria, but also near-universal expression of the DARC-null genotype associated with low neutrophil counts. The mechanisms underlying the interaction between the DARC genotype and white blood cell counts, and how this may influence the function and cross-talk of neutrophils with other immune cells such as T cells, NK cells, and macrophages will form an important part of future research as these new studies provide new avenues in understanding neutrophils and how they may respond to pathogens. Acknowledgements The authors would like to thank Kewreshini Naidoo for assistance with literature scans and Siyabonga Nikwe for assistance with graphic illustrations. Research work in the authors’ laboratory is funded by the South African Research Chairs Initiative of the South African Department of Science and Technology through the National Research Foundation. Additional funding was received from the International AIDS Vaccine
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Myeloid biology
Initiative, the Victor Daitz Chair in HIV/TB Research, and an International Early Career Scientist Award from the Howard Hughes Medical Institute. Conflicts of interest There are no conflicts of interest.
REFERENCES AND RECOMMENDED READING Papers of particular interest, published within the annual period of review, have been highlighted as: & of special interest && of outstanding interest 1. Mestas J, Hughes CC. Of mice and not men: differences between mouse and human immunology. J Immunol 2004; 172:2731–2738. 2. Borregaard N. Neutrophils, from marrow to microbes. Immunity 2010; 33:657–670. 3. Bugl S, Wirths S, Muller MR, et al. Current insights into neutrophil homeostasis. Ann N Y Acad Sci 2012; 1266:171–178. 4. Summers C, Rankin SM, Condliffe AM, et al. Neutrophil kinetics in health and disease. Trends Immunol 2010; 31:318–324. 5. Amulic B, Cazalet C, Hayes GL, et al. Neutrophil function: from mechanisms to && disease. Annu Rev Immunol 2012; 30:459–489. An excellet review summarizing the new functions of neutrophils beyond inflammation as regulators of the immune system. 6. Mocsai A. Diverse novel functions of neutrophils in immunity, inflammation, && and beyond. J Exp Med 2013; 210:1283–1299. Another excellent recent article highlighting the novel and sophisticated functions of neutrophil functions beyond inflammation. 7. Boudaly S. Activation of dendritic cells by polymorphonuclear neutrophils. Front Biosci 2009; 14:1589–1595. 8. Silva MT. Neutrophils and macrophages work in concert as inducers and effectors of adaptive immunity against extracellular and intracellular microbial pathogens. J Leukoc Biol 2010; 87:805–813. 9. Costantini C, Cassatella MA. The defensive alliance between neutrophils and NK cells as a novel arm of innate immunity. J Leukoc Biol 2011; 89:221– 233. 10. Scapini P, Bazzoni F, Cassatella MA. Regulation of B-cell-activating factor (BAFF)/B lymphocyte stimulator (BLyS) expression in human neutrophils. Immunol Lett 2008; 116:1–6. 11. Muller I, Munder M, Kropf P, et al. Polymorphonuclear neutrophils and T lymphocytes: strange bedfellows or brothers in arms? Trends Immunol 2009; 30:522–530. 12. Haddy TB, Rana SR, Castro O. Benign ethnic neutropenia: what is a normal absolute neutrophil count? J Lab Clin Med 1999; 133:15–22. 13. Paz Z, Nails M, Ziv E. The genetics of benign neutropenia. Isr Med Assoc J 2011; 13:625–629. 14. Donadieu J, Fenneteau O, Beaupain B, et al. Congenital neutropenia: diagnosis, molecular bases and patient management. Orphanet J Rare Dis 2011; 6:26. 15. Boxer LA. How to approach neutropenia? Hematology Am Soc Hematol Educ && Program 2012; 2012:174–182. This article enhances the general understanding of different types of neutropenia, cause, and implication in clinical settings. 16. Rezaei N, Moazzami K, Aghamohammadi A, et al. Neutropenia and primary immunodeficiency diseases. Int Rev Immunol 2009; 28:335–366. 17. Sokolic R. Neutropenia in primary immunodeficiency. Curr Opin Hematol & 2013; 20:55–65. The genetic defects associated with congenital neutropenia-associated disorders are summarized in this review. 18. Grann VR, Ziv E, Joseph CK, et al. Duffy (Fy), DARC, and neutropenia among women from the United States, Europe and the Caribbean. Br J Haematol 2008; 143:288–293. 19. Hsieh MM, Everhart JE, Byrd-Holt DD, et al. Prevalence of neutropenia in the U.S. population: age, sex, smoking status, and ethnic differences. Ann Intern Med 2007; 146:486–492. 20. Shoenfeld Y, Ben-Tal O, Berliner S, et al. The outcome of bacterial infection in subjects with benign familial leukopenia (BFL). Biomed Pharmacother 1985; 39:23–26. 21. Hsieh MM, Tisdale JF, Rodgers GP, et al. Neutrophil count in African Americans: lowering the target cutoff to initiate or resume chemotherapy? J Clin Oncol 2010; 28:1633–1637. 22. Grann VR, Bowman N, Joseph C, et al. Neutropenia in 6 ethnic groups from the Caribbean and the U.S. Cancer 2008; 113:854–860. 23. Shaper AG, Lewis P. Genetic neutropenia in people of African origin. Lancet 1971; 2:1021–1023.
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24. Denic S, Showqi S, Klein C, et al. Prevalence, phenotype and inheritance of benign neutropenia in Arabs. BMC Blood Disord 2009; 9:3. 25. Weingarten MA, Pottick-Schwartz EA, Brauner A. The epidemiology of benign leukopenia in Yemenite Jews. Isr J Med Sci 1993; 29:297– 299. 26. Nalls MA, Wilson JG, Patterson NJ, et al. Admixture mapping of white cell count: genetic locus responsible for lower white blood cell count in the Health ABC and Jackson Heart studies. Am J Hum Genet 2008; 82:81–87. 27. Reich D, Nalls MA, Kao WH, et al. Reduced neutrophil count in people of African descent is due to a regulatory variant in the Duffy antigen receptor for chemokines gene. PLoS Genet 2009; 5:e1000360. 28. He W, Neil S, Kulkarni H, et al. Duffy antigen receptor for chemokines mediates trans-infection of HIV-1 from red blood cells to target cells and affects HIV-AIDS susceptibility. Cell Host Microbe 2008; 4:52–62. 29. Julg B, Reddy S, van der Stok M, et al. Lack of Duffy antigen receptor for chemokines: no influence on HIV disease progression in an African treatmentnaive population. Cell Host Microbe 2009; 5:413–415; author reply 8–9. 30. Lee JS, Frevert CW, Wurfel MM, et al. Duffy antigen facilitates movement of chemokine across the endothelium in vitro and promotes neutrophil transmigration in vitro and in vivo. J Immunol 2003; 170:5244–5251. 31. Reiner AP, Lettre G, Nalls MA, et al. Genome-wide association study of white blood cell count in 16 388 African Americans: the Continental Origins and Genetic Epidemiology Network (COGENT). PLoS Genet 2011; 7:e1002108. 32. Crosslin DR, McDavid A, Weston N, et al. Genetic variants associated && with the white blood cell count in 13 923 subjects in the eMERGE Network. Hum Genet 2012; 131:639–652. This study also conducted in large cohorts of European and African Americans confirmed the association of the DARC-null trait to white blood cell counts. In addition, the study identified for the first time that other SNPs previously associated with asthma were also associated with white blood cell counts. 33. Levine AM, Karim R, Mack W, et al. Neutropenia in human immunodeficiency virus infection: data from the women’s interagency HIV study. Arch Intern Med 2006; 166:405–410. 34. Horne KC, Li X, Jacobson LP, et al. Duffy antigen polymorphisms do not alter progression of HIV in African Americans in the MACS cohort. Cell Host Microbe 2009; 5:415–417; author reply 8–9. 35. Walley NM, Julg B, Dickson SP, et al. The Duffy antigen receptor for chemokines null promoter variant does not influence HIV-1 acquisition or disease progression. Cell Host Microbe 2009; 5:408–410; author reply 18–19. 36. Winkler CA, An P, Johnson R, et al. Expression of Duffy antigen receptor for chemokines (DARC) has no effect on HIV-1 acquisition or progression to AIDS in African Americans. Cell Host Microbe 2009; 5:411–413; author reply 8–9. 37. Ramsuran V, Kulkarni H, He W, et al. Duffy-null-associated low neutrophil counts influence HIV-1 susceptibility in high-risk South African black women. Clin Infect Dis 2011; 52:1248–1256. 38. Kourtis AP, Hudgens MG, Kayira D. Neutrophil count in African mothers && and newborns and HIV transmission risk. N Engl J Med 2012; 367:2260– 2262. Higher neutrophil counts in the mother and in newborn were found to be associated with lower risk of mother-to-child HIV transmission in a study conducted in Malawi. 39. Kulkarni H, Marconi VC, He W, et al. The Duffy-null state is associated with a survival advantage in leukopenic HIV-infected persons of African ancestry. Blood 2009; 114:2783–2792. 40. Horuk R, Chitnis CE, Darbonne WC, et al. A receptor for the malarial parasite Plasmodium vivax: the erythrocyte chemokine receptor. Science 1993; 261:1182–1184. 41. Tournamille C, Blancher A, Le Van Kim C, et al. Sequence, evolution and ligand binding properties of mammalian Duffy antigen/receptor for chemokines. Immunogenetics 2004; 55:682–694. 42. Cunnington AJ, Njie M, Correa S, et al. Prolonged neutrophil dysfunction after Plasmodium falciparum malaria is related to hemolysis and heme oxygenase-1 induction. J Immunol 2012; 189:5336–5346. 43. Dey S, Bindu S, Goyal M, et al. Impact of intravascular hemolysis in malaria on liver dysfunction: involvement of hepatic free heme overload, NF-kappaB activation, and neutrophil infiltration. J Biol Chem 2012; 287:26630– 26646. 44. Porcherie A, Mathieu C, Peronet R, et al. Critical role of the neutrophilassociated high-affinity receptor for IgE in the pathogenesis of experimental cerebral malaria. J Exp Med 2011; 208:2225–2236. 45. Walther M, De Caul A, Aka P, et al. HMOX1 gene promoter alleles and high HO-1 levels are associated with severe malaria in Gambian children. PLoS Pathog 2012; 8:e1002579. 46. McMorran BJ, Wieczorski L, Drysdale KE, et al. Platelet factor 4 and Duffy & antigen required for platelet killing of Plasmodium falciparum. Science 2012; 338:1348–1351. DARC receptor essential for binding of platelet factor 4 (PF4) leading to efficient platelet-mediated killing of Plasmodium falciparum. 47. Afenyi-Annan A, Kail M, Combs MR, et al. Lack of Duffy antigen expression is associated with organ damage in patients with sickle cell disease. Transfusion 2008; 48:917–924.
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Neutrophils in Africans Thobakgale and Ndung’u 48. Drasar ER, Menzel S, Fulford T, et al. The effect of Duffy antigen receptor for chemokines on severity in sickle cell disease. Haematologica 2013; 98:e87–e89. 49. Zhang X, Majlessi L, Deriaud E, et al. Coactivation of Syk kinase and MyD88 adaptor protein pathways by bacteria promotes regulatory properties of neutrophils. Immunity 2009; 31:761–771. 50. Yang CT, Cambier CJ, Davis JM, et al. Neutrophils exert protection in the early tuberculous granuloma by oxidative killing of mycobacteria phagocytosed from infected macrophages. Cell Host Microbe 2012; 12:301– 312. Neutrophils contribute to containing mycobacteria infection through engulfment of dying cells within the granuloma rather than by interaction with the microbes.
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51. Martineau AR, Newton SM, Wilkinson KA, et al. Neutrophil-mediated innate immune resistance to mycobacteria. J Clin Invest 2007; 117:1988– 1994.
52. Jenne CN, Wong CH, Zemp FJ, et al. Neutrophils recruited to sites of infection protect from virus challenge by releasing neutrophil extracellular traps. Cell Host Microbe 2013; 13:169–180. A second study to suggest a protective role of neutrophils in innate antiviral immunity. Poxvirus infection led to the recruitment of neutrophils and platelets to the liver sinusoids, forming NETs and in the process protecting host cells from infection. 53. Saitoh T, Komano J, Saitoh Y, et al. Neutrophil extracellular traps mediate & a host defense response to human immunodeficiency virus-1. Cell Host Microbe 2012; 12:109–116. One of the first studies to demonstrate the ability of neutrophils to eliminate HIV and mediate an antiviral response through the formation of NETs. 54. Duffy D, Perrin H, Abadie V, et al. Neutrophils transport antigen from the & dermis to the bone marrow, initiating a source of memory CD8þ T cells. Immunity 2012; 37:917–929. A role of neutrophils in the antigen-specific T-cell response is suggested in mouse models, in which neutrophils are shown to be virus carrier cells from the dermis to the bone marrow initiating a source of memory CD8þ T cells. &
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