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Clinical Science Aspects

X-Linked IRAK1 Polymorphism is Associated with Sex-Related Differences in Polymorphonuclear Granulocyte and Monocyte Activation and Response Variabilities

Qin, Yong; Peña, Geber; Morcillo, Patrick; Singh, Sukhwinder; Mosenthal, Anne C.; Livingston, David H.; Spolarics, Zoltan

Author Information
doi: 10.1097/SHK.0000000000001404

Abstract

INTRODUCTION

Functional variability has been shown to be advantageous during the immune response and after environmental or pathological challenges (1–3) and could be affected by the action of sex hormones (4–10). It has also been proposed that inherent X-linked genetic polymorphisms may be contributing factors in causing sex-related differences in cellular phenotypes and response variabilities (11–14). This nonhormonal hypothesis is based on the differences in sex chromosome homeostasis between males and females. The male Y chromosome carries only a few genes predominantly involved in sex determination with only limited immunomodulatory effects (15). By contrast, the X chromosome (ChrX) is rich in immune-modulatory genes and its regulation is grossly different between the sexes (11–14). Males carry one ChrX passed on from the mother, whereas females carry two ChrXs, one inherited from each parent. Due to the sex-linked inheritance pattern, males with X-linked variant alleles are overrepresented in the population (13, 14). Furthermore, in females, the potential double dose of protein expression from the two ChrXs is compensated by random ChrX inactivation during early embryonic development resulting in cellular mosaicism for the expression of respective parental ChrXs (16). The presence of ChrX mosaicism for common X-linked genetic polymorphisms in females and the lack of this condition in males may result in sex-biased differences in cellular phenotypes (11–14).

In this study, we tested this hypothesis in the context of the X-linked polymorphic IRAK1 gene. The variant IRAK1 haplotype is widespread across ethnic and racial groups ranging between 20% and 60% population frequencies (17–21). Within the variant IRAK1 haplotype, the critical mutation is a nonsynonymous single nucleotide variant (rs1059703), which results in a T to C base change at 1674 with a consequent replacement of leucine with serine at amino acid 532 in the kinase domain of IRAK1 resulting in increased kinase activity. This allelic variant of the IRAK1 haplotype is in linkage disequilibrium with the other exonic nonsynonymous allele (rs1059702) and other multiple intronic alleles in most individuals (19, 21). The amino acid changes are associated with increased phosphorylation of IRAK1 and subsequent augmentation of TLR-mediated NFkB activation (19).

Despite the fact that this common variant IRAK1 was shown to be associated with markedly worsened clinical outcomes after sepsis or trauma by independent studies (19–22), the phenotypic cellular effects caused by this polymorphism are less known (19). Thus, testing the impact of this common X-linked polymorphism on ChrX skewing, PMN and monocyte activation and their response variabilities (23–25) may provide insights into the immunomodulatory mechanisms associated with sex-related differences in clinical outcomes.

In this study, using flow cytometry, we determined the expression of a selected set of membrane proteins associated with myeloid cell activation, signaling, cell adhesion, and transmigration (26–33) under baseline (nonstimulated) conditions as well as after ex vivo cell stimulations by lipopolysaccharide and phorbol ester. Cell responses and their inter- and intrasubject variabilities were compared after stratification by sex alone and/or IRAK1 genotypes. Furthermore, because female X-linked cellular mosaicism may also contribute to increased functional heterogeneity of WBCs (23–25) and trauma was shown to induce de novo changes in ChrX skewing (14, 34), we also determined the effect of variant-IRAK1 on ChrX inactivation ratios (XCI) in trauma patients.

We found that grouping by sex alone did not show marked differences in PMN and monocyte activation or inter- or intrasubject cell response variabilities. However, stratification by the IRAK1 genotype revealed augmented cell activation in variant-IRAK1 subjects, which was accompanied by decreased intrasubject cell response variabilities. Variant IRAK1 also suppressed injury-induced de novo ChrX skewing in trauma patients during the clinical course.

MATERIALS AND METHODS

Human subjects

The healthy cohort represented consecutive consenting volunteers recruited at Rutgers Biomedical Health Sciences, Newark, NJ (convenience sampling). A total of 51 subjects (25 males 26 females) were enrolled. Mean age (±SD) was 29 ± 7 (M) and 28 ± 7 (F). Racial/ethnic distribution according to self-identification was 6 African American, 2 Hispanic, 12 Caucasian, 18 Asian, and 13 undisclosed/unknown or mixed. IRAK1 genotyping for the critical variant allele (rs1059703) showed 10 variant and 15 WT hemizygous males; and 8 homozygous variant, 8 heterozygous mosaic, and 10 WT females.

The trauma cohort represented 201 consecutive female patients admitted to the trauma service at the New Jersey Trauma Center at University Hospital in Newark, Level I Trauma Center. Inclusion criteria were females with heterogeneity for the Androgen Receptor at the HUMARA locus; age more than 18 years; Injury Severity Score (ISS) at least 9; Glasgow Coma Score (GCS) more than 3; and admission to the Progressive Care or Surgical Intensive Care Units. Patients who expired within 24 h of injury were excluded. Racial/ethnic distribution of the trauma cohort was 85 African American, 32 Hispanics, 45 Caucasian, 6 Asian, and 33 undisclosed/unknown or mixed. Age distribution stratified by the IRAK1 genotype (rs1059703) was 49 ± 20 (WT), 52 ± 21 (variant), and 47 ± 21 (heterozygous).

Informed consent was obtained from contributing volunteers, but was waived for the trauma sample analyses because only routinely collected “ready to discard” leftover samples were used, and personal clinical files were not accessed. The study was approved by the Institutional Review Board of Rutgers New Jersey Medical School.

Blood collection

Blood from healthy volunteers was drawn into commercial, EDTA containing vacutainer tubes and aliquoted for same-day cell incubations and flow cytometry as well as subsequent DNA analyses. DNA analyses of trauma samples were performed on the leftover, ready to discard blood drawn at admission and as available from subsequent routine tests.

DNA isolation and genotyping

DNA from whole blood was isolated using QIAamp mini kit (QIAGEN). The IRAK1 sequence flanking the variant allele (rs1059703) was amplified using primers 5’-CCTCTTTTCCACTTGCAGGTGTAC (forward) and 5’-GTGCTGGACACGTAGGAGTTCTC (reverse). Amplification was carried out as 3 min at 95°C, followed by 35 cycles of 15 s at 95°C, 15 s at 60°C, 2 s at 72°C and then final extension at 72°C. The PCR product was purified by QIAquick as per manufacturer's instructions and genotype was determined by sequencing in a Seqstudio Genetic Analyzer (Thermo Fisher Scientific). The other exonic variant allele of the IRAK1 haplotype (rs1059702) was assayed by the TaqMan Genotyping Assay kit (Thermo Fisher Scientific) using a 7500 Real-time PCR system as per manufacturer's instructions and the following amplification cycles: 1 min at 60°C, 10 min at 95°C, followed by 40 cycles of 15 s at 95°C, 1 min at 60°C and then final extension at 60°C. As expected, the two exonic variant alleles of IRAK1 were in strong linkage disequilibrium (86% of the cohort had both alleles simultaneously).

Cell incubation and flow cytometry

Fresh whole blood was incubated in the absence (vehicle control) or presence of lipopolysaccharide (100 ng/mL ultrapure-LPS, from Escherichia coli 055:B5; InvivoGen) plus phorbol 12-myristate 13-acetate (PMA, 80 nM, Sigma) for 30 min at 37oC. Following the incubations, a 0.1 mL aliquot was taken and incubated for 30 min at RT in the dark with anti-Human conjugated antibodies against CD11b/Mac-1, CD45, CD14, CD63, and CD66b (fluorochromes, clones, and catalog numbers are listed in supplemental Table 2S, Supplemental Digital Content, https://links.lww.com/SHK/A910). Samples were processed using the manufacturer-provided protocol and reagents (BD Biosciences). At the end of the incubation, cells were fixed, and acquisition was performed in a multicolor BD LSR II Flow Cytometer (BD Biosciences) within an hour after fixation. A total of 50,000 events of the CD45+ WBCs were collected, and PMN and monocytes gated by Side Scatter (SSC-A) and CD14 positivity (for gating strategy and typical findings, see supplemental Fig. 1S, Supplemental Digital Content, https://links.lww.com/SHK/A910). Gated PMNs and monocytes represented on average 61% and 6% of CD45+ cells, respectively (supplemental Table 1S, Supplemental Digital Content, https://links.lww.com/SHK/A910). Cells were analyzed for mean fluorescence intensity. Cell response variability within subjects was assessed by the robust coefficient of variance (%rCV) (25) as determined the Flow Cytometer (BD LSR II, BD Biosciences). For the determination of ChrX skewing in different major WBC types, PMN and monocytes were stained as described above, whereas B cells and T cells were acquired by CD45CD3 and CD45CD18 positivity. Cells were sorted using FACSARIA II cytometer. Sorted cells were sedimented by centrifugation and subjected to DNA analyses.

Determination of ChrX skewing (XCI)

Assay conditions were described in detail previously (34). Briefly, DNA analyses were performed on the leftover, ready to discard blood using QIAamp mini kit (QIAGEN). XCI ratios were determined by measuring DNA methylation at the androgen receptor locus (HUMARA) (35, 36) after digesting DNA with 10U of methylation-sensitive and insensitive restriction endonucleases HpaII and RsaI, respectively. The analysis and PCR amplifications were performed according to a previously published procedure (36) using fluorescent labeled primers: 5’-FAM/TCCAGA ATCTGTTCCAGAGCGTGC, and 5’-V-GCTGTGAAGGTTGCTGTTCCTCAT for PCR amplification. PCR-amplified fragments were electrophoresed on Seqstudio Genetic Analyzer and quantified by the area under the curve for each allele peak, using Gene-Mapper software (Thermo Fisher Scientific) (37).

Statistical analyses

Statistical calculations were performed using JMP software (SAS Institute, Cary, NC). For continuous variables, we used ANOVA followed by t test for pairwise or Tukey–Kramer for multiple comparisons. Differences in intersubject variability were assessed by the Levene test. Differences in means between samples with unequal variance were assessed by the Welch test. Differences in intrasubject cell response variabilities were tested by comparing the robust coefficient of variance (%rCV) of cell response distributions (25) as determined the Flow Cytometer (BD LSR II, BD Biosciences). %rCV was used because it is more resistant to outlier events (BD FACSDiva, BD Biosciences). Differences in %rCV were tested by t test/nonparametric Wilcoxon-rank-sum test. Statistically significant difference was concluded at P < 0.05.

RESULTS

Routine hematological parameters in males and females of the healthy cohort are shown in Table 1. As expected, RBC count, hemoglobin concentration, and hematocrit were significantly lower in females than males. PMN, lymphocyte, monocyte, and platelet counts were similar in males and females. Testing for unequal intersubject variances showed no differences for RBCs, platelets, and monocytes, but variances for PMN and lymphocyte counts were greater in females than males.

T1
Table 1:
Blood cell counts in the volunteer sample stratified by sex

Next, we determined the expression of a selected set of membrane proteins associated with cell activation using flow cytometry. Mean fluorescence intensity (MFI) was determined, which corresponds with the membrane expression of the target proteins. A typical finding is shown in supplemental Fig. 1S, Supplemental Digital Content, https://links.lww.com/SHK/A910. LPS/PMA stimulation resulted in a marked statistically significant increase in the expression of CD11b and CD66b in PMNs which was similar in males and females (Fig. 1, A and G). Stratification by IRAK1 genotype (male–female data merged, heterozygous mosaic females excluded) showed a greater increase of CD11b and CD66b expression in variant-IRAK1 subjects as compared with WT (Fig. 1, B and H). Testing for differences in PMN activation after grouping by all genotypes did not reveal major response differences between variant-IRAK1 males and females, whereas heterozygous mosaic females showed an intermediate response (Fig. 1, C and I).

F1
Fig. 1:
IRAK1 haplotype augments membrane expression of CD11b and CD66b in activated PMNs but blunts intrasubject response variabilities.

Cell activation also resulted in a marked and similar increases in the membrane expression of CD45 and CD63 in PMNs in males and females. The differences in CD45 and CD63 expression between WT and variant-IRAK1 subjects did not reach statistically significant levels (data not shown). Nonstimulated PMNs showed no remarkable differences in the membrane expression of these markers after any groupings (data not shown).

Intrasubject cell response variabilities of activated PMNs (%rCV) were similar in males and females (Fig. 1, D and J); however, subjects with the variant-IRAK1 showed decreased PMN response variabilities for CD11b and CD66b expression in activated cells (Fig. 1, E and K). Stratification by genotype showed decreased variability in PMN responses of variant-IRAK1 subjects independent of sex, whereas heterozygous females presented intermediate variability (Fig. 1, F and L). %rCV of nonstimulated PMNs showed no remarkable differences after any groupings (data not shown).

Stimulation of monocytes resulted in a marked increase in the expression of CD11b and CD63 with similar responses in males and females (Fig. 2, A and G). Similar to the observations on PMNs, monocytes carrying the variant-IRAK1 gene showed a greater increase of CD11b and CD63 expressions as compared with WT (Fig. 2, B and H). Testing for differences in cell activation after grouping by all genotypes showed a more pronounced response in variant-IRAK1 males than females (Fig. 2, C and I). CD11b expression was also increased in females with cellular mosaicism for variant/WT IRAK1 as compared with WT females (Fig. 2C).

F2
Fig. 2:
IRAK1 haplotype augments membrane expression of CD11b and CD63 in activated monocytes but blunts intrasubject response variabilities.

Ex vivo monocyte activation also resulted in a marked and similar increases in the membrane expression of CD45 and CD14 in males or females. The differences in CD45 and CD14 expression between WT and variant-IRAK1 subjects did not reach statistically significant levels (data not shown). Nonstimulated monocytes showed no statistically significant differences in the membrane expression of these markers after any groupings (data not shown).

Activated monocytes showed increased CD11b intrasubject response variability in males versus females, but CD63 response variability was similar (Fig. 2, D and J). In accordance with the observations on PMNs, activated monocytes from variant-IRAK1 subjects showed decreased intrasubject variability as compared with WT (Fig. 2, E and K). Stratification by IRAK1 genotypes also showed decreased intrasubject response variability of monocytes from variant-IRAK1 subjects with a more consistent effects in males (Fig. 2, F and L). %rCV of nonstimulated monocytes showed no remarkable differences after any groupings (data not shown).

ChrX mosaicism for multiple nonpathological X-linked polymorphisms may increase cellular variability exclusively in females. This nonpathological cellular heterogeneity may modulate cell trafficking during trauma resulting in acute de novo ChrX skewing as we published previously (14, 34). The variant IRAK1 haplotype, however, represents a risk allele (19–22) and, as shown here, causes augmented cell activation that may impact cell trafficking during the trauma course. Therefore, we tested how variant IRAK1 affects the trauma-induced changes in ChrX inactivation (XCI) ratios. XCI ratios were similar in WT, mosaic, and variant-IRAK1 WBCs from healthy females (Fig. 3A). Because the degree and direction of ChrX skewing may be different between myeloid and lymphoid white blood cells, we also determined XCI ratios after sorting WBCs by flow cytometry from three individuals of each genotype (Fig. 3B). There were only small differences in the degree of skewing between myeloid and lymphoid cells, and, importantly, the “direction” of skewing was always the same (i.e., no switch toward XCI < 1 in any cells; Fig. 3B).

F3
Fig. 3:
Patients homozygous for variant IRAK1 or heterozygous mosaics for the variant/WT allele present depressed trauma-induced de novo ChrX skewing as compared to WT trauma subjects.

Next, we tested the effect of variant IRAK1on XCI-ratios in mixed WBCs in trauma patients. Variant IRAK1 and heterozygous mosaicism for the WT/variant alleles showed lower mean XCI ratio at admission, but the difference did not reach statistical significance (Fig. 3C). The mean trauma-induced de novo XCI ratio change during the clinical course was decreased in variant-IRAK1 and heterozygous mosaic patients as compared with WT (Fig. 3D). Importantly, when XCI-ratio change was expressed as percent change over initial within each individual trauma subject, the blunting effect on ChrX skewing was also evident in variant homozygous IRAK1 as well as heterozygous mosaic IRAK1 patients as compared with WT (Fig. 3E).

DISCUSSION

Although the association of the variant IRAK1 haplotype with poor outcomes from trauma, sepsis, and autoimmune conditions is well established (18–22, 38, 39), the phenotypic changes in cellular functions associated with IRAK1 polymorphism are less studied. Here we report that the variant IRAK1 haplotype results in an augmented increase in the expression of membrane proteins involved in PMN and monocyte activation and cell trafficking. Augmented expression of these adhesion molecules may differently affect cell migration of mosaic subsets and could also modulate ChrX skewing.

CD11b in concert with CD66b are critical protein components of integrins participating in a variety of granulocyte functions, including signaling, cell adhesion, transmigration, and chemotaxis (26, 28–30, 32, 33). CD63 may also form complexes with integrins including CD11b and participates in cell adhesion and vesicle trafficking (27, 32, 33). The augmented membrane expression of these proteins in variant-IRAK1 cells is consistent with previous findings showing increased NFκB levels in WBCs from septic variant-IRAK1 subjects (19). These observations, together with previous animal studies, which indicated survival advantage in IRAK-1 deficiency (40, 41), suggest that an increased proinflammatory response is likely to be a part of pathology leading to the deleterious effects of the IRAK1 haplotype in sepsis and trauma (19–22).

The observations also indicate that although the increased cell activation in variant-IRAK1 subjects is accompanied by similar (CD66b and CD63) or increased (CD11b) intersubject response variabilities in PMNs and monocytes, the intrasubject response variability is consistently and similarly decreased in variant-IRAK1 cells. This observation suggests that despite the greater potential range of cell responses in variant-IRAK1 subjects, the increased cell activation potential caused by the variant allele narrows the cellular response-range within individuals (Fig. 4). This blunted variability may affect cellular adaptation to the uneven tissue milieu or dynamically changing pathophysiological conditions observed in sepsis or after injuries and thereby could contribute to the pathology of the IRAK1 haplotype (19, 21, 22, 38).

F4
Fig. 4:
Proposed schema indicating that variant IRAK1 augments intersubject and decreases intrasubject response variabilities of PMNs and monocytes.

The fact that when comparing all genotypes, the augmented PMN and monocyte activation and their blunted response variabilities were more consistent and pronounced in variant-IRAK1 males suggests that interactions between the IRAK1 genetic background and sex hormones modulate the eventual cellular phenotype. However, a larger sample size is needed to substantiate the potential importance of a more pronounced cell-activating effects in variant-IRAK1 males.

We proposed previously that female X-linked cellular mosaicism may contribute to increased functional heterogeneity of WBCs, which, in turn, may be an immunomodulatory mechanism unique to females (13, 14). We also demonstrated that female trauma patients present dynamic changes in XCI ratios during the clinical course of trauma (14, 34). Changing XCI ratios of circulating WBCs in response to trauma is most likely the reflection of phenotypic differences between mosaic WBC populations driven by polymorphic differences in their respective parental ChrXs affecting cell adhesion, migration, apoptosis, or necrosis. Here we demonstrate that female trauma patients who are homo- or heterozygous for the variant IRAK1 present blunted ChrX skewing during the clinical course of injury. This suggests that the increased cell activation caused by variant-IRAK1 masks the effects of other modulatory, otherwise nonpathological, X-linked polymorphisms, which normally would allow manifesting differences in cell trafficking and associated ChrX skewing.

It was a somewhat surprising finding that heterozygous IRAK1 trauma subjects also showed blunted ChrX skewing considering that the presence of the two mosaic cell populations is expected to cause a widening rather than narrowing of ChrX skewing. This observation suggests that activation signals were conveyed from variant to WT cells through paracrine mechanisms consistent with similar conclusions from animal studies (41, 42). It remains to be determined how cellular mosaicism for variant/WT IRAK1 alleles affects the clinical outcome in sepsis or trauma, as this has not been evaluated in previous clinical studies (19, 21, 22, 38).

The observation that spontaneous ChrX skewing similarly affected all major WBC types in healthy individuals (Fig. 3AB) indicates that random and spontaneous skewing of mosaic subpopulations occurs at the stem cell level before commitment toward the major myeloid or lymphoid lines in the bone marrow. However, cell trafficking, life span, apoptosis, and necrosis associated with de novo ChrX skewing, which may manifest during sepsis and trauma, could affect myeloid and lymphoid cells differently. Thus, the blunting effect of variant IRAK1 observed here on mixed WBCs from trauma patients is most likely the result of a combination of different degrees of ChrX skewing on myeloid and lymphoid cells. However, because circulating PMN shows the most marked kinetics in blood together with depleted lymphocyte counts during the trauma course (14), neutrophils are the likely cellular candidates resulting in blunted ChrX skewing in variant-IRAK1 trauma patients.

The fact that variant and heterozygous IRAK1 trauma patients already showed a trend toward blunted XCI-ratios at admission suggests that IRAK1 may affect baseline ChrX skewing at the level of the bone marrow. However, it is more likely that trafficking of PMNs was already affected at some degree during the time period between the time of injury and first blood sampling at the hospital, which is about 30 min on average. Nevertheless, the finding that variant IRAK1 showed no skewing in the small healthy sample, together with the fact that ChrX skewing was also blunted during the hospital course (Fig. 3, D and E) clearly indicates that variant IRAK1 blunts de novo ChrX skewing. The potential role of blunted ChrX skewing in contributing to a worsened outcome of variant-IRAK1 septic and trauma (19–22) patients remains to be elucidated in future studies.

It is noteworthy that we did not find any remarkable differences in cell activation and inter- or intrasubject cell response variability when directly comparing healthy males and females in our sample. By contrast, differences in cell activation and response variabilities were clearly and consistently affected in variant-IRAK1 subjects of the same cohort. However, as the variant IRAK1 is X-linked and its inheritance follows an X-linked pattern, the observations may be relevant in the broader context of sex-based outcome differences (43). Our healthy cohort was generated by convenience sampling of 51 consecutive individuals and was not controlled for racial or ethnic background; therefore, it did not generate a genotype distribution reflective of the general population. However, in large cohorts which are more representative of the general population, the X-linked genotype pattern may manifest sex-related differences. For example, in a Caucasian or African cohort with a 25% allele frequency of variant-IRAK1, the genotype distributions would approximate 6% affected homozygous females versus 25% affected hemizygous males (4-fold difference). In an Asian cohort, where the reported variant-IRAK1 allele frequency is in the 50% to 70% range (17, 18, 20, 38, 44), the number of affected hemizygous variant males would be about double than that of affected homozygous females. These facts together with our current observations highlight potential limitations of studies testing sex-based differences in immune responses by simply comparing males and females without evaluating the potential confounding effects of common polymorphic X-linked risk alleles.

A limitation of the study is that basophils, eosinophils, and dendritic cells or other monocyte subsets of circulating myeloid cells were not targeted and analyzed individually. However, the dominant component of granulocytes is neutrophils, whereas basophils and eosinophils represent less than 2% and 7% of granulocytes, and likewise, dendritic cells present less than 1% of circulating mononuclear myeloid cells. Thus, although variant-IRAK1 granulocyte or monocyte subsets may respond at different degrees, it is unlikely that the selective response of these subsets accounted for the observed differences in this study.

Taken together, our study reveals that direct comparison between males and females rarely show differences in the degree of activation of PMNs and monocytes or their inter- or intrasubject response variabilities. However, augmented cell activation together with decreased intrasubject response variabilities readily manifest in the variant-IRAK. Upon in vivo cell activation, as produced by trauma, a diminished de novo ChrX skewing in variant-IRAK1 females may also affect the response variability of white blood cells. These differences in cellular phenotypes between WT and variant IRAK1 subjects may be contributing factors affecting the course of sepsis and trauma and may also impact sex-based outcome differences due to the X-linked inheritance pattern and high prevalence of the IRAK1 haplotype.

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Keywords:

Chromosome X; inactivation; mosaicism; polymorphism; skewing; trauma; variability; white blood cells

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