Sex-Differential Impact of Human Cytomegalovirus Infection on In Vitro Reactivity to Toll-Like Receptor 2, 4 and 7/8 Stimulation in Gambian Infants
by
, ,
Momodou Cox
1,2
,
Jane U. Adetifa
1,
Fatou Noho-Konteh
1,
Lady C. Sanyang
1,
Abdoulie Drammeh
1,
Magdalena Plebanski
2,3
,
Hilton C. Whittle
1,4,
Sarah L. Rowland-Jones
1,5,
Iain Robertson
6 and
Katie L. Flanagan
1,2,6,*
1
Infant Immunology Group, Vaccines and Immunity Theme, MRC Unit, P.O. Box 273, Fajara, The Gambia
2
School of Health & Biomedical Science, RMIT University, Melbourne, VIC 3083, Australia
3
Department of Immunology and Pathology, Monash University, Melbourne, VIC 3004, Australia
4
Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1E 7HT, UK
5
Nuffield Department of Medicine, University of Oxford, Oxford OX3 9DU, UK
6
School of Medicine and School of Health Sciences, University of Tasmania, Launceston, Tasmania 7250, Australia
*
Author to whom correspondence should be addressed.
Vaccines 2020, 8(3), 407; https://doi.org/10.3390/vaccines8030407
Submission received: 12 June 2020
/
Revised: 14 July 2020
/
Accepted: 20 July 2020
/
Published: 22 July 2020
(This article belongs to the Special Issue Research on Innate Immunity and Inflammation)
Abstract
:Human cytomegalovirus (HCMV) infection rates approach 100% by the first year of life in low-income countries. It is not known if this drives changes to innate immunity in early life and thereby altered immune reactivity to infections and vaccines. Given the panoply of sex differences in immunity, it is feasible that any immunological effects of HCMV would differ in males and females. We analysed ex vivo innate cytokine responses to a panel of toll-like receptor (TLR) ligands in 108 nine-month-old Gambian males and females participating in a vaccine trial. We found evidence that HCMV suppressed reactivity to TLR2 and TLR7/8 stimulation in females but not males. This is likely to contribute to sex differences in responses to infections and vaccines in early life and has implications for the development of TLR ligands as vaccine adjuvants. Development of an effective HCMV vaccine would be able to circumvent some of these potentially negative effects of HCMV infection in childhood.
1. Introduction
The human herpes virus 5, more commonly known as human cytomegalovirus (HCMV), is known to infect a high proportion of children in low-income settings in the first year of life [1]. Acutely infected individuals go on to develop a chronic latent infection that is generally asymptomatic, although it can reactivate throughout life, particularly during times of immune suppression [2]. Congenital HCMV infection leads to a series of neurodevelopmental defects including deafness and cognitive impairment [3]. While infection after the neonatal period is not thought to lead to these serious outcomes, the clinical and immunological implications of establishing HCMV infection in early life remain uncertain.
Infection with HCMV activates the production of antiviral type I interferons (IFNs) by dendritic cells (DCs) [4]. While TLR2 recognizes HCMV, TLR2 does not appear to be responsible for triggering an antiviral type 1 IFN response [5,6]. Indeed, MCMV studies suggest that toll-like receptors (TLR) 3, 7 and 9 act redundantly to promote the early initial type I interferon, although TLR9 probably plays a greater role and TLR2 signalling may also be involved [7]. The dendritic cell (DC)-derived type I IFNs activate NF-κB via intracellular signalling pathways, leading to the expression of multiple IFN-responsive genes with antiviral properties and the production of innate pro-inflammatory cytokines including TNF-α, IL-1β and IL-6 [8]. Type I IFNs also promote activation of natural killer (NK) cell cytotoxicity and NK and natural killer T (NKT) cell IFN-γ production. Gamma delta (γδ) T cells, which have both innate and adaptive properties, are also thought to play a protective role against HCMV infection [9,10].
Despite the activation of an antiviral response, HCMV establishes latent infection by residing in monocyte precursors and tissue stromal cells which undergo constant host immune surveillance to prevent reactivation of infection [11]. While it is the adaptive immune system that is responsible for immune surveillance and long-term control of viral replication to maintain a state of latent infection [11,12], early in infection the innate immune system and NK cell activation are also thought to play a critical role [7,9,10]. Cytomegalovirus has co-evolved with its vertebrate hosts for over 100 million years to become a master manipulator of host immunity in order to maintain long-term asymptomatic latent infection [13,14]. These involve multiple immune escape strategies to avoid both innate and adaptive immunity [14], resulting in what has been termed “mutually assured survival”. This involves the expression of host protein homologues including chemokines and a range of immunomodulatory proteins unique to HCMV, including proteins that can evade type I IFN signalling [15] and mechanisms to avoid NK cell-mediated effector functions [7,16]. The CMV immediate-early (IE) gene expresses nuclear phosphoproteins that act as transcriptional regulators involved in evasion of innate immunity and reactivation from latency [17]. The immunosuppressive cytokine IL-10 also plays a role in latent infection, and MCMV expresses an IL-10 orthologue gene during the latent phase [18]. Unfortunately, much of the MCMV evidence cannot be extrapolated to humans since it derives from murine models of infection which have unique immune-modulatory genes that are not present in HCMV [19].
We now understand that the innate immune system has memory, a process that has been called “trained immunity,” which operates largely via epigenetic effects [20]. It is therefore not surprising that HCMV has been shown to have long-lasting adaptive effects on innate immunity, including epigenetic modification of NK cells [21]. This NK cell training impacts the innate response not only to CMV [22] but potentially to other immune challenges. There is a need for research to understand how asymptomatic latent HCMV infection impacts ability to respond to challenge by diverse pathogens and vaccination. This is particularly pertinent to high disease burden settings such as Africa, where HCMV infection rates are almost 100% by one year of age [1] and where multiple vaccinations are administered to HCMV+ infants.
Sex differences in susceptibility and outcomes from many infections are well described, including viruses [23], and females seem to have a greater HCMV seroprevalence in most studies, suggesting greater susceptibility [24]. Immunologically, sex differences have been shown for several aspects of HCMV influences on adaptive immunity, including circulating memory T cell subsets [25] and T cell cytokine profiles [26]. One study hints at sex differences in the impact of HCMV on circulating NK cell frequencies, with males having higher CD56dimNK cells (mature NK cells) than females [27], but otherwise, sex-differential effects of HCMV infection on innate immunity have not been described.
While the effects of HCMV on the innate immune system have therefore been characterised in some detail, mostly in animal models, the consequences of asymptomatic latent HCMV infection in early life on innate reactivity to immunological challenge in vivo is not known. It is also not known whether biological sex would have an influence on any observed effects in prepubertal children. We therefore selected a panel of TLR ligands to interrogate the ex vivo innate cytokine response in African infant males and females participating in a vaccine trial [28]. An investigation of the impact of HCMV on TLR reactivity is particularly timely since TLR ligands, particularly TLR7/8 agonists, are being developed as adjuvants for infant vaccines [29]. These will be employed in infants in low-income settings with very high rates of infant HCMV infection.
2. Materials and Methods
2.1. Study Population
The 9-month old infants in this study were participating in a vaccine trial, the results of which have been published previously [28,30]. They were healthy afebrile male and female children recruited at the Sukuta Health Centre near the coast of The Gambia in West Africa. All study children were well on the day of sample collection and had received all recommended vaccines except for two-thirds of the children who had only received 2 diphtheria-tetanus-whole cell pertussis (DTP) doses as required in the study protocol.
2.2. Ethics Statement
The study was approved by the Joint Gambia Government/MRC Ethics Committee (project number SCC1085) and the London School of Hygiene and Tropical Medicine Ethics Committee. Written/thumb-printed informed consent was provided by the parent/guardian of the child in accordance for the Declaration of Helsinki.
2.3. Diagnosis of HCMV Infection
Urine samples collected at 9 months of age were tested for the presence of HCMV DNA by a nested polymerase chain reaction (PCR) with a cut off of approximately 25 DNA copies/mL, as described previously [31,32]. Serum HCMV IgG was not analysed since it is unreliable in this age group due to the potential for false-positive results from maternally derived antibody. A negative control known to contain no HCMV was used in every PCR run, and positive controls of 250 and 2500 DNA copies/mL were also included to reduce the likelihood of false-positive diagnoses. Urine was not available at 9 months for all infants in the study cohort, and only those with a urine HCMV result are included.
2.4. Whole Blood Cultures and Cytokine Multiplex Analysis
A heparinised whole venous blood sample was taken at 9 months of age prior to administration of the study vaccines. Whole blood was cultured in 100 μL aliquots in 96-well U-bottom plates with a panel of TLR ligands: heat-killed listeria monocytogenes (HKLM) (109 cells/mL) (TLR2 agonist), Escherichia coli K12 lipopolysaccharide protein S (LPS) (1 μg/mL) (TLR4 agonist), flagellin (10 μg/mL) (TLR5 agonist) and CLO75 (10 μg/mL) (TLR7/8 agonist) (all from InvivoGen, San Diego, CA, USA). The TLR ligand concentrations used were selected as optimal in prior titration assays in infants and are in line with our previous published studies in this age group [33]. RPMI alone was used in the unstimulated control wells. Plates were incubated for 16 h at 37 °C, 5% CO2, centrifuged at 2000 rpm for 5 min and 50 μL supernatant collected and stored at −20 °C. The Bio-Plex 200 Suspension Array system (Bio-Rad, Hercules, CA, USA) was used to analyse cytokines according to the manufacturer’s instructions (Bio-Rad, Belgium). A 5-plex array (IL-1β, IL-6, IL-10, IL-12(p70), TNF-α) was used. Innate assays were only performed for those infants in the study with sufficient blood volume and quality, and those with low volume, clotted or contaminated blood samples were excluded.
2.5. Statistical Analysis
General linear modelling was used to estimate the geometric mean, geometric standard deviation (GSD), mean difference, 95% confidence interval and p-value for each variable for infants with and without HCMV infection. The effects of infant sex were determined by estimating HCMV:sex interactions. Analysis was performed on natural logarithmic transformed cytokine levels and then converted back to natural numbers by exponential transformation; thus, the GSD is a multiplicative factor and not an absolute value. In some cases, there was insufficient blood: it clotted or wells became contaminated; thus, cytokine values were missing for 95 (3.5%) of the potential 2700 wells in the study (108 infants tested for 5 cytokines to 5 conditions (unstimulated and 4 TLR ligands)). These missing data were substituted by multiple imputation (using “mi estimate: glm” Stata syntax, with 50 imputations). Multivariate linear regression modelling was used in order to capture the potential inter-relationships between the immune parameters tested. For this, infant sex, HCMV status and all other cytokine/chemokine values were considered potential covariates for adjustment. Covariates selected by forward stepwise regression had an entry p-value of 0.15 and a removal p-value of 0.2 and were presented to the statistical model as z-scores of the natural logarithms of the raw values. The z-scores used for the adjustment process were the subject value minus the mean value divided by the standard deviation. The adjusted values therefore control for the responses to all the other TLR agonists. Unadjusted and adjusted p-values are shown in the tables. All statistical analyses were performed using Stata MP2 V16.0 (StataCorp, College Station, TX, USA).
3. Results
3.1. Infant Characteristics
Data from 108 study infants who had both urine HCMV and innate cytokine data at 9 months of age are included in the analysis. Seventy-three (67.6%) of the 108 infants were HCMV-positive (HCMV+) at 9 months of age, and 35 (32.4%) were HCMV-negative (HCMV-) (Table 1). There was an approximately equal number of males and females in the analysis, which included 55 (50.9% of participants) males and 53 (49.1% of participants) females; 66% of males were HCMV+, and 69.1% of females were HCMV+ (Table 1). All samples were collected during the dry season, which runs from November to May in The Gambia. All children were well and afebrile on the day of blood collection; we therefore did not test for any other pathogens such as malaria.
3.2. Minimal Effects of HCMV Infection on TLR Ligand Responses When Males and Females Are Analysed Together
When all infants were analysed together, the only TLR agonist/cytokine combination affected by HCMV infection was a lower TNF-α to HKLM stimulation in the HCMV infected as compared to the uninfected group (p = 0.04 unadjusted, 0.066 adjusted) (Table 2). IL-1β was also lower in unstimulated control wells in infants infected with HCMV as compared to the uninfected infants (p = 0.81 unadjusted, 0.022 adjusted) (Table 3). IL-6, IL-12 (p70), and IL-10 responses in the TLR ligand cultures were not affected by HCMV status when all infants were analysed together (Table 4, Table 5 and Table 6). The TNF-α:IL-10 pro-:anti-inflammatory cytokine ratio was also not affected by HCMV status when analysing all infants combined (Table 7).
3.3. Evidence for Effects of HCMV Infection on TLR Ligand Responses in Females but Not Males
TNF-α and TNF-α:IL-10 responses to the TLR2 ligand HKLM were lower in HCMV+ females as compared to the HCMV- females (TNF-α: unadjusted p = 0.021, adjusted p = 0.022; TNF-α:IL-10: unadjusted p = 0.038, adjusted p = 0.021) (Figure 1A), whereas there was no apparent effect of HCMV infection in females or males on TNF-α responses to the other three TLR ligands—LPS, flagellin or CLO75 (Table 2 and Table 7).
HCMV+ females had lower IL-6 reactivity to the TLR7/8 ligand CLO75 as compared to their HCMV- counterparts (unadjusted p = 0.014, adjusted p = 0.008), while HCMV+ males had greater CLO75-stimulated IL-6 responses as compared to the HCMV- males (unadjusted p = 0.15, adjusted p = 0.018) (Figure 1B, Table 4). A comparison of the effect of HCMV effect in males and females showed a highly significant difference between the sexes with respect to CLO75 IL-6 responses (unadjusted p = 0.005, adjusted p < 0.001) (Figure 1B, Table 4).
By contrast, IL-10 responses to CLO75 were higher in HCMV+ females as compared to HCMV- females (unadjusted p = 0.47, adjusted p = 0.005), while there was no such effect of HCMV status in the male group (Figure 1C, Table 5). The HCMV effect was significantly different between males and females for CLO75-stimulated IL-10 production (p = 0.016) (Figure 1C, Table 5). IL-10 responses to the TLR4 ligand LPS were also significantly different when comparing the HCMV effect in males and females (unadjusted p = 0.040, adjusted p = 0.034) (Figure 1D). This is because HCMV+ females had higher IL-10 than the HCMV- females, while HCMV+ males had the reciprocal effect of lower IL-10 than their HCMV- counterparts, albeit the differences were not significant in the separate sex analyses (Figure 1D, Table 5).
The only TLR ligand responses not affected by HCMV status were cytokine responses to the TLR5 agonist flagellin. Furthermore, HCMV status had no effect on IL-1β or IL-12 production to the panel of TLR ligands.
4. Discussion
The effect of HCMV infection on innate reactivity has not been investigated in infants in areas of the world where infection rates reach almost 100% by one year of age [1]. Furthermore, the possibility that effects might differ in the sexes has not been investigated. Herein, we show that in the non-sex-stratified analysis, HCMV infection had little impact on in vitro innate cytokine responses to a panel consisting of TLR2, 4, 5 and 7/8 ligands, except for reduced TNF-α reactivity to TLR2 stimulation with HKLM.
Sex differences in susceptibility to infections and responses to vaccination are evident from birth, with males being the more susceptible to a range of infections and more likely to succumb to sepsis, while females have more robust antibody responses to many vaccines [23,34,35]. On the few occasions that it has been examined, infant males have been shown to have greater innate immune responses to vaccination as compared to females [35]. The potential reasons for these immunological sex differences include sex differences in circulating sex hormone levels, sex differences in the microbiota and sex differences in the expression of the multiple immune response genes expressed on the X chromosome [23,34,35]. When males and females were analysed separately in our study, we found that HCMV+ females had reduced TNF-α and TNF-α:IL-10 to HKLM stimulation, but there was no such effect of HCMV infection in males. Females also had reduced IL-6 and increased IL-10 to CLO75 stimulation. By contrast, HCMV+ males had increased IL-6 to CLO75 stimulation. TNF-α and IL-6 are classic pro-inflammatory cytokines [36], while IL-10 has suppressive immunoregulatory properties [37]. Therefore, our results suggest that HCMV infection suppresses innate reactivity to TLR2 and TLR7/8 stimulation in infant females but not in males. This could compromise female reactivity to pathogens recognised via TLR2 and TLR7/8 receptors, which include a range of Gram-positive and Gram-negative bacteria, viruses, yeasts, mycoplasma and parasites [38,39].
These findings have potentially important implications given the high incidence of infant HCMV infection in low-income settings. If HCMV infection suppresses innate immunity, particularly in females, this could alter female susceptibility to the plethora of infections to which they are exposed in early life. Furthermore, vaccine immunogenicity relies on an early innate response, which is intrinsically linked to the adaptive immune response [40]. Suppressed innate immunity in HCMV+ females could therefore result in poorer innate and adaptive immune responses to vaccination. Indeed, our recent study found that HCMV+ females had lower tetanus toxoid and pertactin IgG titres following diphtheria–tetanus–whole-cell-pertussis immunisation as compared to their HCMV- counterparts, although only on unadjusted analysis [41]. Having said that, most infants achieved seroprotective levels regardless of HCMV status in this study, suggesting limited clinical impact of HCMV infection [41]. In the same study, HCMV+ infants had suppressed overall T cell immunity, as evidenced by lower cytokine responses to stimulation with anti-CD3/anti-CD28 [41]. Overall, this suggests that HCMV infection can suppress both innate and adaptive immunity in infants in high-HCMV-incidence settings, consistent with its well-described evolutionary mechanisms aimed at avoiding host immunity.
Several limitations of this study should be borne in mind. The relatively small number of infants in the HCMV- groups reduces the power of the study. Another issue is that some of the HCMV- infants may have been infected earlier in life but no longer shedding the virus in their urine and thus classified as HCMV-. However, previous studies in Gambian infants show that those infected in the first year of life continue to shed virus in urine for many months [1,42], so we do not think that many infants would have been misclassified. Future longitudinal studies would help confirm whether the phenotype we describe is specific to active shedders or all infants who have acquired HCMV infection. The whole blood assay (WBA) used in this study is considered a more physiological assay, representative of true in vivo reactivity, compared to using separated peripheral blood mononuclear cells or other purified cell populations. A variety of cells in whole blood contribute to the cytokine responses to TLR stimulation, including activated monocytes, macrophages, dendritic cells, NK cells, NK T cell, γδ T cells and neutrophils. Therefore, a limitation of this approach is that we do not know precisely which cells are producing the cytokines measured.
It is interesting that responses to the TLR7/8 ligand CLO75 are particularly affected by co-existing HCMV infection in our study. Newborns have a robust response to this TLR ligand from birth [33,43], and as a result, it is being developed as an adjuvant for infant vaccines, particularly for neonates [29,44,45]. Our results suggest that HCMV+ infant females may have impaired reactivity to this adjuvant approach.
5. Conclusions
In summary, we show for the first time that early-life HCMV infection can impact innate immunity, in infant females in particular, with potential implications for their ability to respond to infections and vaccines. The clinical implications are uncertain, but further contribute to our understanding of the well-described sex differences in disease susceptibility and vaccine responses in the prepubertal period and lend further weight to the arguments for developing an effective HCMV vaccine.
Author Contributions
Conceptualization, H.C.W., K.L.F., M.P. and S.L.R.-J.; methodology, A.D., F.N-K., K.L.F., J.U.A., L.C.S. and M.C.; supervision, K.L.F.; validation, K.L.F., H.C.W. and S.L.R.-J.; formal analysis, I.R.; data curation, F.N.-K. and K.F.; writing—original draft preparation, K.L.F. and M.C.; writing—review and editing, A.D., F.N.-K., H.C.W., I.R., J.U.A., K.L.F., L.C.S., M.C., M.P. and S.L.R.-J.; funding acquisition, H.C.W., K.L.F. and S.L.R-J. All authors have read and agreed to the published version of the manuscript.
Funding
This study was funded by a UK Medical Research Council strategic grant (grant number G0701291) and MRC Gambia core funds (grant number SCC1085). Statistical analysis was supported by the Clifford Craig Foundation, Tasmania, Australia.
Acknowledgments
We would like to sincerely thank the children and their parents/guardians who agreed to participate in this study. We also thank all the staff at Sukuta Health Centre, in particular Ebrima Touray and Sally Savage. We also thank Sarah Burl for laboratory management.
Conflicts of Interest
The authors have no conflict of interest to declare.
References
- Miles, D.J.; van der Sande, M.; Jeffries, D.; Kaye, S.; Ismaili, J.; Ojuola, O.; Sanneh, M.; Touray, E.S.; Waight, P.; Rowland-Jones, S.; et al. Cytomegalovirus infection in gambian infants leads to profound CD8 T-Cell differentiation. J. Virol. 2007, 81, 5766–5776. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Forte, E.; Zhang, Z.; Thorp, E.B.; Hummel, M. Cytomegalovirus latency and reactivation: An intricate interplay with the host immune response. Front. Cell Infect. Microbiol. 2020, 10, 130. [Google Scholar] [CrossRef] [PubMed]
- Cheeran, M.C.; James, R.; Lokensgard, J.R.; Schleiss, M.R. Neuropathogenesis of congenital cytomegalovirus infection: Disease mechanisms and prospects for intervention. Clin. Microbiol. Rev. 2009, 22, 99–126. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Van de Berg, P.J.; Heutinck, K.M.; Raabe, R.; Minnee, R.C.; La Young, S.; van Donselaar-van der Pant, K.A.; Bemelman, F.J.; van Lier, R.A.; ten Berge, I.J. Human cytomegalovirus induces systemic immune activation characterized by a Type 1 Cytokine Signature. J. Infect. Dis. 2010, 202, 690–699. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Compton, T.; Kurt-Jones, E.A.; Boehme, K.W.; Belko, J.; Latz, E.; Golenbock, D.T.; Finberg, R.W. Human cytomegalovirus activates inflammatory cytokine responses via CD14 and toll-like receptor 2. J. Virol. 2003, 77, 4588–4596. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Juckem, L.K.; Boehme, K.W.; Feire, A.L.; Compton, T. Differential initiation of innate immune responses induced by human cytomegalovirus entry into fibroblast cells. J. Immunol. 2008, 180, 4965–4977. [Google Scholar] [CrossRef] [Green Version]
- Loewendorf, A.; Benedict, C.A. Modulation of host innate and adaptive immune defenses by Cytomegalovirus: Timing is everything. J. Intern. Med. 2010, 267, 483–501. [Google Scholar] [CrossRef]
- Jun Cui, J.; Chen, Y.; Wang, H.Y.; Wang, R.F. Mechanisms and pathways of innate immune activation and regulation in health and cancer. Hum. Vaccin. Immunother. 2014, 10, 3270–3285. [Google Scholar] [CrossRef] [Green Version]
- Terrazzini, N.; Kern, F. Cell-mediated immunity to human CMV infection: A brief overview. F1000Prime Rep. 2014, 6, 28. [Google Scholar] [CrossRef]
- Camille Khairallah, C.; Julie Déchanet-Merville, J.; Capone, M. γδ T Cell-mediated immunity to cytomegalovirus infection. Front. Immunol. 2017, 8, 105. [Google Scholar] [CrossRef] [Green Version]
- Seckert, C.K.; Griessl, M.; Büttner, J.K.; Scheller, S.; Simon, C.O.; Kropp, K.A.; Renzaho, A.; Kühnapfel, B.; Grzimek, N.K.; Reddehase, M.J. Viral latency drives ‘Memory inflation’: A unifying hypothesis linking two hallmarks of Cytomegalovirus infection. Med. Microbiol. Immunol. 2012, 201, 551–566. [Google Scholar] [CrossRef] [PubMed]
- Slobedman, B.; Mocarski, E.S. Mechanisms modulating immune clearance during human cytomegalovirus latency. Proc. Natl. Acad. Sci. USA 2012, 109, 14291–14292. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Reddehase, M.J. ‘Checks and balances’ in cytomegalovirus-host cohabitation. Med. Microbiol. Immunol. 2019, 208, 259–261. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Miller-Kittrell, M.; Sparer, T.E. Feeling manipulated: Cytomegalovirus immune manipulation. Virol. J. 2009, 6, 4. [Google Scholar] [CrossRef] [Green Version]
- Paulus, C.; Krauss, S.; Nevels, M. A human cytomegalovirus antagonist of type I IFN-dependent signal transducer and activator of transcription signaling. Proc. Natl. Acad. Sci. USA 2006, 103, 3840–3845. [Google Scholar] [CrossRef] [Green Version]
- Goodier, M.R.; Jonjić, S.; Eleanor, M.; Riley, E.M.; Juranić Lisnić, V. CMV and Natural killer cells: Shaping the response to vaccination. Eur. J. Immunol. 2018, 48, 50–65. [Google Scholar] [CrossRef]
- Christina Paulus, C.; Nevels, M. The human cytomegalovirus major immediate-early proteins as antagonists of intrinsic and innate antiviral host responses. Viruses 2009, 1, 760–779. [Google Scholar] [CrossRef] [Green Version]
- Cheung, A.K.; Gottlieb, D.J.; Plachter, B.; Pepperl-Klindworth, S.; Avdic, S.; Cunningham, A.L.; Abendroth, A.; Slobedman, B. The role of the human Cytomegalovirus UL111A gene in Down-Regulating CD4+ T-cell recognition of latently infected cells: Implications for virus elimination during latency. Blood 2009, 114, 4128–4137. [Google Scholar] [CrossRef] [Green Version]
- Roark, H.K.; Jenks, J.A.; Permar, S.R.; Schleiss, M.R. Animal models of congenital Cytomegalovirus transmission: Implications for vaccine development. J. Infect. Dis. 2020, 221, S60–S73. [Google Scholar] [CrossRef] [Green Version]
- Netea, M.G.; Joosten, L.A.; Latz, E.; Mills, K.H.; Natoli, G.; Stunnenberg, H.G.; O‘Neill, L.A.; Xavier, R.J. Trained immunity: A program of innate immune memory in health and disease. Science 2016, 352. [Google Scholar] [CrossRef] [Green Version]
- Luetke-Eversloh, M.; Quirin Hammer, Q.; Pawel Durek, P.; Karl Nordström, K.; Gasparoni, G.; Pink, M.; Hamann, A.; Walter, J.; Chang, H.D.; Dong, J.; et al. Human Cytomegalovirus drives epigenetic imprinting of the IFNG locus in NKG2Chi natural killer cells. PLoS Pathog. 2014, 10. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Hammer, Q.; Romagnani, C. About training and memory: NK-Cell adaptation to viral infections. Adv. Immunol. 2017, 133, 171–207. [Google Scholar] [CrossRef] [PubMed]
- Klein, S.L.; Flanagan, K.L. Sex differences in immune responses. Nat. Rev. Immunol. 2016, 16, 626–638. [Google Scholar] [CrossRef] [PubMed]
- Cannon, M.J.; Schmid, S.D.; Hyde, T.B. Review of Cytomegalovirus seroprevalence and demographic characteristics associated with infection. Rev. Med. Virol. 2010, 20, 202–213. [Google Scholar] [CrossRef] [PubMed]
- Di Benedetto, S.; Derhovanessian, E.; Steinhagen-Thiessen, E.; Goldeck, D.; Müller, L.; Pawelec, G. Impact of age, sex and CMV-infection on peripheral T cell phenotypes: Results from the Berlin BASE-II Study. Biogerontology 2015, 16, 631–643. [Google Scholar] [CrossRef]
- Villacres, M.C.; Longmate, J.; Auge, C.; Diamond, D.J. Predominant Type 1 CMV-specific memory T-helper response in humans: Evidence for gender differences in cytokine secretion. Hum. Immunol. 2004, 65, 476–485. [Google Scholar] [CrossRef]
- Reed, R.G.; Al-Attar, A.; Presnell, S.R.; Lutz, C.T.; Suzanne, C.; Segerstrom, S.C. A longitudinal study of the stability, variability, and interdependencies among late-differentiated T and NK cell subsets in older adults. Exp. Gerontol. 2019, 121, 46–54. [Google Scholar] [CrossRef]
- Noho-Konteh, F.; Adetifa, J.U.; Cox, M.; Hossin, S.; Reynolds, J.; Le, M.T.; Sanyang, L.C.; Drammeh, A.; Plebanski, M.; Forster, T.; et al. Sex-differential non-vaccine-specific immunological effects of diphtheria-tetanus-pertussis and measles vaccination. Clin. Infect. Dis. 2016, 63, 1213–1226. [Google Scholar] [CrossRef] [Green Version]
- Dowling, D.J.; Tan, Z.; Prokopowicz, Z.M.; Palmer, C.D.; Maura-Ann, H.; Matthews, M.A.H.; Dietsch, G.N.; Hershberg, R.M.; Levy, O. The ultra-potent and selective TLR8 agonist VTX-294 activates human newborn and adult leukocytes. PLoS ONE 2013, 8. [Google Scholar] [CrossRef] [Green Version]
- Ndure, J.; Noho-Konteh, F.; Adetifa, J.U.; Cox, M.; Barker, F.; Le, M.T.; Sanyang, L.C.; Drammeh, A.; Whittle, H.C.; Clarke, E.; et al. Negative correlation between circulating CD4(+)FOXP3(+)CD127(-) regulatory T cells and subsequent antibody responses to infant measles vaccine but not diphtheria-tetanus-pertussis vaccine implies a regulatory role. Front. Immunol. 2017, 8, 921. [Google Scholar] [CrossRef] [Green Version]
- Preiser, W.; Brauninger, S.; Schwerdtfeger, R.; Ayliffe, U.; Garson, J.A.; Brink, N.S.; Franck, S.; Doerr, H.W.; Rabenau, H.F. Evaluation of diagnostic methods for the detection of cytomegalovirus in recipients of allogeneic stem cell transplants. J. Clin. Virol. 2001, 20, 59–70. [Google Scholar] [CrossRef]
- Marchant, A.; Appay, V.; van der Sande, M.; Dulphy, N.; Liesnard, C.; Kidd, M.; Kaye, S.; Ojuola, O.; Gillespie, G.M.; Vargas Cuero, A.L.; et al. Mature CD8(+) T lymphocyte response to viral infection during fetal life. J. Clin. Investig. 2003, 111, 1747–1755. [Google Scholar] [CrossRef] [PubMed]
- Burl, S.; Townend, J.; Njie-Jobe, J.; Cox, M.; Adetifa, U.J.; Touray, E.; Philbin, V.J.; Mancuso, C.; Kampmann, B.; Whittle, H.; et al. Age-dependent maturation of Toll-like receptor-mediated cytokine responses in Gambian infants. PLoS ONE 2011, 6. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Flanagan, K.L.; Fink, A.L.; Plebanski, M.; Klein, S.L. Sex and gender differences in the outcomes of vaccination over the life course. Ann. Rev. Cell. Dev. Biol. 2017, 33, 577–599. [Google Scholar] [CrossRef] [PubMed]
- Harper, A.; Flanagan, K.L. Effect of sex on vaccination outcomes: Important but frequently overlooked. Curr. Opin. Pharmacol. 2018, 41, 122–127. [Google Scholar] [CrossRef] [PubMed]
- Feghali, C.A.; Wright, T.M. Cytokines in acute and chronic inflammation. Front. Biosci. 1997, 2, d12–d26. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Moore, K.W.; de Waal Malefyt, R.; Coffman, R.L.; O‘Garra, A. Interleukin-10 and the interleukin-10 Receptor. Annu. Rev. Immunol. 2001, 19, 683–765. [Google Scholar] [CrossRef]
- Kawai, T.; Akira, S. Toll-like receptors and their crosstalk with other innate receptors in infection and immunity. Immunity 2011, 34, 637–650. [Google Scholar] [CrossRef] [Green Version]
- Kumar, H.; Kawai, T.; Akira, S. Pathogen recognition by the innate immune system. Int. Rev. Immunol. 2011, 30, 16–34. [Google Scholar] [CrossRef]
- Fitzgerald, K.A.; Kagan, J.C. Toll-like receptors and the control of immunity. Cell 2020, 180, 1044–1066. [Google Scholar] [CrossRef]
- Cox, M.; Adetifa, J.U.; Noho-Konteh, F.; Njie-Jobe, J.; Sanyang, L.C.; Drammeh, A.; Plebanski, M.; Whittle, H.C.; Rowland-Jones, S.L.; Robertson, I.; et al. Limited impact of human cytomegalovirus infection in African infants on vaccine-specific responses following diphtheria-tetanus-pertussis and measles vaccination. Front. Immunol. 2020, 11, 1083. [Google Scholar] [CrossRef] [PubMed]
- Bello, C.; Whittle, H. Cytomegalovirus infection in Gambian mothers and their babies. J. Clin. Pathol. 1991, 44, 366–369. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Reikie, B.A.; Adams, R.C.; Ruck, C.E.; Ho, K.; Leligdowicz, A.; Pillay, S.; Naidoo, S.; Fortuno, E.S., 3rd; de Beer, C.; Preiser, W.; et al. Ontogeny of toll-like receptor mediated cytokine responses of South African infants throughout the first year of life. PLoS ONE 2012, 7. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Dowling, D.J.; Scott, E.A.; Scheid, A.; Bergelson, I.; Joshi, S.; Pietrasanta, C.; Brightman, S.; Sanchez-Schmitz, G.; van Haren, S.D.; Ninković, J.; et al. Toll-like receptor 8 agonist nanoparticles mimic immunomodulating effects of the live BCG vaccine and enhance neonatal innate and adaptive immune responses. J. Allergy Clin. Immunol. 2017, 140, 1339–1350. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Dowling, D.J.; van Haren, S.D.; Scheid, A.; Bergelson, I.; Kim, D.; Mancuso, C.J.; Foppen, W.; Ozonoff, A.; Fresh, L.; Theriot, T.B. TLR7/8 adjuvant overcomes newborn hyporesponsiveness to pneumococcal conjugate vaccine at birth. JCI Insight 2017, 2. [Google Scholar] [CrossRef] [PubMed] [Green Version]
Figure 1.
The significant sex-differential cytokine responses in the study. Whole heparinized blood was stimulated for 16 h with TLR ligands, and cytokine concentrations were measured in culture supernatants by multiplex assay. (A). HCMV+ females had lower TNF-α responses to TLR2 (HKLM) stimulation than HCMV- females. (B). HCMV+ females had lower IL-6 responses to TLR7/8 (CLO75) stimulation than HCMV- females, while males had the opposite pattern of higher responses in the HCMV+ group. There was a significant difference in IL-6 responses to CLO75 between males and females. (C). HCMV+ females had greater IL-10 production to CLO75 than HCMV- females, whereas HCMV+ and HCMV- males had comparable CLO75-stimulated IL-10. There was a significant difference in IL-10 responses to CLO75 between males and females. (D). There was no significant difference in IL-10 responses to TLR4 (LPS) stimulation between HCMV+ and HCMV- infants for males or females, but comparing male and female responses did show a significant difference. The individual symbols equal individual infants and the bar shows the geometric mean value in pg/mL. N/S = not significant, M = males and F = females.
Figure 1.
The significant sex-differential cytokine responses in the study. Whole heparinized blood was stimulated for 16 h with TLR ligands, and cytokine concentrations were measured in culture supernatants by multiplex assay. (A). HCMV+ females had lower TNF-α responses to TLR2 (HKLM) stimulation than HCMV- females. (B). HCMV+ females had lower IL-6 responses to TLR7/8 (CLO75) stimulation than HCMV- females, while males had the opposite pattern of higher responses in the HCMV+ group. There was a significant difference in IL-6 responses to CLO75 between males and females. (C). HCMV+ females had greater IL-10 production to CLO75 than HCMV- females, whereas HCMV+ and HCMV- males had comparable CLO75-stimulated IL-10. There was a significant difference in IL-10 responses to CLO75 between males and females. (D). There was no significant difference in IL-10 responses to TLR4 (LPS) stimulation between HCMV+ and HCMV- infants for males or females, but comparing male and female responses did show a significant difference. The individual symbols equal individual infants and the bar shows the geometric mean value in pg/mL. N/S = not significant, M = males and F = females.
Table 1.
Infant numbers according to sex and HCMV status.
| Group | HCMV+ | HCMV- | Total |
|---|---|---|---|
| All Infants | 73 (67.6%) | 35 (32.4%) | 108 |
| Males | 35 (66.0%) | 18 (34.0%) | 53 |
| Females | 38 (69.1%) | 17 (30.9%) | 55 |
Number of infants shown with % of total in brackets.
Table 2.
Effect of HCMV infection on TLR ligand-stimulated TNF-α responses by infant sex.
| TNF-α | HCMV− | HCMV+ | Comparison (Unadjusted) | Comparison (Adjusted) | ||||||
|---|---|---|---|---|---|---|---|---|---|---|
| N | Geo Mean (GSD) | N | Geo Mean (GSD) | Δ | 95% CI | p-Value | Δ | 95% CI | p-Value | |
| Unstimulated | ||||||||||
| ALL | 34 | 73 (9.0) | 73 | 97 (9.5) | 25 | (−33 to 165) | 0.52 | 426 | (−88 to 1144) | 0.12 |
| Male | 17 | 139 (9.7) | 35 | 111 (9.5) | −27 | (−109 to 278) | 0.75 | 588 | (−56 to 1581) | 0.08 |
| Female | 17 | 38 (7.2) | 38 | 89 (9.8) | 51 | (−10 to 238) | 0.15 | 272 | (−1874 to 1503) | 0.54 |
| M vs. F | −78 | (−170 to 446) | 0.23 | 316 | (−585 to 2092) | 0.51 | ||||
| TLR2 (HKLM) | ||||||||||
| ALL | 35 | 15,529 (3.2) | 73 | 8818 (5.3) | −6711 | (−10,386 to −409) | 0.040 | −1369 | (−2414 to 107) | 0.066 |
| Male | 18 | 12,050 (4.2) | 35 | 7843 (4.4) | −4207 | (−8630 to 5937) | 0.31 | −338 | (−1712 to 1966) | 0.72 |
| Female | 17 | 20,311 (2.0) | 38 | 9614 (6.1) | −10,698 | (−15,214 to −2178) | 0.021 | −2738 | (−7615 to −497) | 0.022 |
| M vs. F | 6490 | (−1406 to 20,944) | 0.55 | 2400 | (−679 to 5880) | 0.21 | ||||
| TLR4 (LPS) | ||||||||||
| ALL | 32 | 6349 (5.0) | 72 | 4661 (5.4) | −1688 | (−3978 to 2816) | 0.37 | −181 | (−1108 to 1254) | 0.76 |
| Male | 16 | 6115 (4.2) | 34 | 5027 (4.5) | −1088 | (−4019 to 5941) | 0.66 | 27 | (−1150 to 2222) | 0.97 |
| Female | 16 | 6591 (6.2) | 38 | 4429 (6.0) | −2161 | (−4996 to 5709) | 0.45 | −396 | (−3837 to 1839) | 0.66 |
| M vs. F | 1073 | (−2644 to 15,336) | 0.77 | 422 | (−1047 to 3909) | 0.74 | ||||
| TLR5 (Flagellin) | ||||||||||
| ALL | 33 | 5245 (4.3) | 69 | 4405 (6.1) | −840 | (−2948 to 3202) | 0.60 | −239 | (−945 to 875) | 0.62 |
| Male | 17 | 5137 (4.6) | 34 | 4326 (5.4) | −811 | (−3429 to 5824) | 0.72 | 151 | (−749 to 1848) | 0.80 |
| Female | 16 | 5362 (4.2) | 35 | 4462 (6.8) | −900 | (−3553 to 5645) | 0.69 | −690 | (−3257 to 895) | 0.32 |
| M vs. F | 89 | (−3053 to 11,572) | 0.99 | 842 | (−247 to 3360) | 0.37 | ||||
| TLR7/8 (CLO75) | ||||||||||
| ALL | 32 | 3960 (3.5) | 68 | 3523 (6.7) | −438 | (−2072 to 2610) | 0.71 | −130 | (−798 to 830) | 0.76 |
| Male | 16 | 3420 (4.6) | 34 | 2678 (4.8) | −742 | (−2358 to 3331) | 0.60 | −164 | (−916 to 1123) | 0.75 |
| Female | 16 | 4586 (2.5) | 34 | 4319 (8.3) | −267 | (−2628 to 4943) | 0.88 | −74 | (−2341 to 1649) | 0.91 |
| M vs. F | −475 | (−2360 to 5890) | 0.77 | −91 | (−1054 to 1978) | 0.88 | ||||
TNF-α levels were analysed in culture supernatants from whole blood stimulated for 16 h with toll-like receptor (TLR) ligands. The table shows geometric mean (geo mean) plus geometric standard deviation (GSD) cytokine levels in pg/mL. The mean difference (Δ; 95% confidence intervals; p-values) between those geometric means were estimated using general linear modelling (unadjusted and adjusted for covariates) with missing data substituted by multiple imputation. The difference between the effect of Human cytomegalovirus (HCMV) in males compared with females was estimated (M vs. F). N = number of infants analysed for each TLR ligand. Bold are the significant effects.
Table 3.
Effect of HCMV infection on TLR ligand-stimulated IL-1β responses by infant sex.
| IL-1β | HCMV− | HCMV+ | Comparison (Unadjusted) | Comparison (Adjusted) | ||||||
|---|---|---|---|---|---|---|---|---|---|---|
| N | Geo Mean (GSD) | N | Geo Mean (GSD) | Δ | 95%CI | p-Value | Δ | 95%CI | p-Value | |
| Unstimulated | ||||||||||
| ALL | 34 | 83 (13.2) | 73 | 73 (11.8) | −10 | (−57 to 120) | 0.81 | −1223 | (−1939 to −212) | 0.022 |
| Male | 17 | 212 (14.4) | 35 | 86 (14.4) | −126 | (−194 to 194) | 0.25 | −1460 | (−2463 to 329) | 0.094 |
| Female | 17 | 32 (8.7) | 38 | 64 (10.4) | 32 | (−13 to 187) | 0.27 | −971 | (−4292 to 415) | 0.15 |
| M vs. F | −159 | (−232 to 376) | 0.11 | −489 | (−1693 to 2056) | 0.65 | ||||
| TLR2 (HKLM) | ||||||||||
| ALL | 35 | 14,290 (3.4) | 73 | 10,137 (7.0) | −4154 | (−8757 to 4280) | 0.27 | 97 | (−1256 to 2168) | 0.91 |
| Male | 18 | 13,556 (3.7) | 35 | 8436 (4.9) | −5120 | (−9800 to 5390) | 0.25 | −379 | (−1729 to 1869) | 0.68 |
| Female | 17 | 15,111 (3.3) | 38 | 11,646 (8.9) | −3464 | (−10,213 to 12,584) | 0.56 | 630 | (−5053 to 4807) | 0.65 |
| M vs. F | −1655 | (−7513 to 17,510) | 0.72 | −1009 | (−2943 to 3511) | 0.54 | ||||
| TLR4 (LPS) | ||||||||||
| ALL | 32 | 5668 (5.7) | 72 | 4038 (6.7) | −1630 | (−3744 to 2809) | 0.37 | −275 | (−1063 to 1021) | 0.61 |
| Male | 16 | 5280 (3.2) | 34 | 4519 (4.9) | −761 | (−3252 to 4789) | 0.70 | 137 | (−830 to 1985) | 0.83 |
| Female | 16 | 6084 (9.0) | 38 | 3737 (8.3) | −2348 | (−4987 to 6637) | 0.44 | −717 | (−4625 to 1314) | 0.39 |
| M vs. F | 1587 | (−1887 to 16,599) | 0.66 | 854 | (−370 to 3944) | 0.43 | ||||
| TLR5 (Flagellin) | ||||||||||
| ALL | 33 | 5063 (4.1) | 69 | 4024 (4.4) | −1039 | (−2832 to 2194) | 0.45 | −491 | (−1146 to 468) | 0.27 |
| Male | 17 | 6032 (4.0) | 34 | 3994 (3.4) | −2037 | (−4206 to 2706) | 0.30 | −897 | (−1665 to 345) | 0.13 |
| Female | 16 | 4204 (4.2) | 35 | 4046 (5.2) | −158 | (−2487 to 5332) | 0.93 | −132 | (−2266 to 1409) | 0.83 |
| M vs. F | −1880 | (−4623 to 6879) | 0.53 | −765 | (−1768 to 1240) | 0.38 | ||||
| TLR7/8 (CLO75) | ||||||||||
| ALL | 32 | 3940 (4.2) | 68 | 3014 (6.5) | −926 | (−2389 to 1917) | 0.43 | −348 | (−812 to 307) | 0.26 |
| Male | 16 | 3536 (6.0) | 34 | 2898 (4.0) | −638 | (−2456 to 4246) | 0.69 | −228 | (−904 to 884) | 0.63 |
| Female | 16 | 4390 (2.8) | 34 | 3105 (8.9) | −1284 | (−3053 to 2823) | 0.42 | −468 | (−2054 to 481) | 0.28 |
| M vs. F | 647 | (−1460 to 8365) | 0.82 | 240 | (−638 to 2029) | 0.69 | ||||
IL-1β levels were analysed in culture supernatants from whole blood stimulated for 16 h with TLR ligands. The table shows geometric mean (geo mean) plus geometric standard deviation (GSD) cytokine levels in pg/mL. The mean difference (Δ; 95% confidence intervals; p-values) between those geometric means was estimated using general linear modelling (unadjusted and adjusted for covariates) with missing data substituted by multiple imputation. The difference between the effect of HCMV in males compared with females was estimated (M vs. F). N = number of infants analysed for each TLR ligand.
Table 4.
Effect of HCMV infection on TLR ligand-stimulated IL-6 responses by infant sex.
| IL-6 | HCMV− | HCMV+ | Comparison (Unadjusted) | Comparison (Adjusted) | ||||||
|---|---|---|---|---|---|---|---|---|---|---|
| N | Geo Mean (GSD) | N | Geo Mean (GSD) | Δ | 95%CI | p-Value | Δ | 95%CI | p-Value | |
| Unstimulated | ||||||||||
| ALL | 34 | 2188 (10.0) | 73 | 2480 (7.2) | 293 | (−1167 to 3839) | 0.78 | 6906 | (−2431 to 21,524) | 0.17 |
| Male | 17 | 5791 (8.5) | 35 | 3312 (7.3) | −2480 | (−4810 to 5388) | 0.37 | 2717 | (−11,543 to 30,714) | 0.78 |
| Female | 17 | 826 (8.0) | 38 | 2027 (7.0) | 1201 | (−166 to 5401) | 0.12 | 8854 | (−11,251 to 26,477) | 0.091 |
| M vs. F | −3681 | (−6360 to 10,339) | 0.085 | −6137 | (−23,124 to 34,746) | 0.37 | ||||
| TLR2 (HKLM) | ||||||||||
| ALL | 35 | 99,711 (2.6) | 73 | 73,393 (3.2) | −26,317 | (−51,057 to 11,003) | 0.14 | −8868 | (−26,981 to 17,855) | 0.46 |
| Male | 18 | 93,368 (2.9) | 35 | 88,146 (3.1) | −5221 | (−45,992 to 70,635) | 0.86 | 6634 | (−22,721 to 58,030) | 0.72 |
| Female | 17 | 10,6898 (2.3) | 38 | 64,113 (3.3) | −42,785 | (−69,160 to 2024) | 0.06 | −23,230 | (−96,836 to 10,025) | 0.14 |
| M vs. F | 37,564 | (−11,617 to 14,8820) | 0.28 | 29,863 | (−6245 to 10,6282) | 0.20 | ||||
| TLR4 (LPS) | ||||||||||
| ALL | 32 | 98,179 (4.4) | 72 | 61,846 (2.9) | −36,334 | (−62,872 to 10,152) | 0.11 | −17,692 | (−35,345 to 10,443) | 0.18 |
| Male | 16 | 11,4889 (2.7) | 34 | 75,326 (2.4) | −39,563 | (−72,343 to 18,475) | 0.15 | −18,329 | (−42,013 to 23,017) | 0.32 |
| Female | 16 | 83,899 (6.3) | 38 | 54,144 (3.2) | −29,755 | (−62,884 to 55,601) | 0.36 | −17,274 | (−10,0509 to 25,908) | 0.34 |
| M vs. F | −9808 | (−60,196 to 14,2396) | 0.98 | −1055 | (−33,561 to 77,520) | 0.89 | ||||
| TLR5 (Flagellin) | ||||||||||
| ALL | 33 | 71,286 (3.6) | 69 | 69,451 (2.8) | −1836 | (−28,974 to 42,709) | 0.92 | 861 | (−16,017 to 27,511) | 0.94 |
| Male | 17 | 77,027 (3.1) | 34 | 72,256 (2.7) | −4771 | (−38,656 to 59,038) | 0.84 | −73 | (−20,553 to 35,652) | 1.00 |
| Female | 16 | 65,655 (4.3) | 35 | 67,485 (2.9) | 1830 | (−34,427 to 80,182) | 0.94 | 1670 | (−58,715 to 47,904) | 0.92 |
| M vs. F | −6602 | (−52,201 to 11,7002) | 0.86 | −1743 | (−30,217 to 68,278) | 0.93 | ||||
| TLR7/8 (CLO75) | ||||||||||
| ALL | 32 | 87,048 (4.1) | 68 | 61,996 (4.3) | −25,052 | (−52,802 to 25,183) | 0.26 | −8595 | (−26,326 to 20,587) | 0.49 |
| Male | 16 | 50,778 (3.2) | 34 | 82,605 (2.8) | 31,827 | (−8476 to 11,0530) | 0.15 | 28,847 | (3689 to 72,477) | 0.018 |
| Female | 16 | 14,9225 (4.3) | 34 | 50,082 (5.3) | −99,143 | (−12,8262 to −29,577) | 0.014 | −57,211 | (−15,7017 to −20,296) | 0.008 |
| M vs. F | 13,0971 | (75,908 to 29,6114) | 0.005 | 86,058 | (50,886 to 17,2231) | <0.001 | ||||
IL-6 levels were analysed in culture supernatants from whole blood stimulated for 16 h with TLR ligands. The table shows geometric mean (geo mean) plus geometric standard deviation (GSD) cytokine levels in pg/mL. The mean difference (Δ; 95% confidence intervals; p-values) between those geometric means were estimated using general linear modelling (unadjusted and adjusted for covariates) with missing data substituted by multiple imputation. The difference between the effect of HCMV in males compared with females was estimated (M vs. F). N = number of infants analysed for each TLR ligand. Bold are the significant effects.
Table 5.
Effect of HCMV infection TLR on ligand-stimulated IL-12(p70) responses by infant sex.
| IL-12 | HCMV− | HCMV+ | Comparison (Unadjusted) | Comparison (Adjusted) | ||||||
|---|---|---|---|---|---|---|---|---|---|---|
| N | Geo Mean (GSD) | N | Geo Mean (GSD) | Δ | 95% CI | p-Value | Δ | 95% CI | p-Value | |
| Unstimulated | ||||||||||
| ALL | 34 | 25 (3.9) | 73 | 25 (6.1) | 0 | (−11 to 22) | 0.95 | −19 | (−60 to 50) | 0.52 |
| Male | 17 | 40 (3.1) | 35 | 27 (6.5) | −13 | (−28 to 22) | 0.35 | −51 | (−101 to 48) | 0.24 |
| Female | 17 | 16 (4.3) | 38 | 25 (6.0) | 9 | (−5 to 42) | 0.30 | 5 | (−166 to 113) | 0.89 |
| M vs. F | −22 | (−41 to 40) | 0.17 | −56 | (−120 to 114) | 0.36 | ||||
| TLR2 (HKLM) | ||||||||||
| ALL | 35 | 117 (2.1) | 73 | 116 (2.7) | −1 | (−35 to 45) | 0.95 | 7 | (−9 to 28) | 0.41 |
| Male | 18 | 121 (2.2) | 35 | 115 (2.1) | −6 | (−47 to 56) | 0.82 | 11 | (−14 to 45) | 0.44 |
| Female | 17 | 113 (2.1) | 38 | 117 (3.2) | 3 | (−42 to 78) | 0.91 | 4 | (−71 to 35) | 0.74 |
| M vs. F | −9 | (−65 to 98) | 0.81 | 6 | (−26 to 59) | 0.75 | ||||
| TLR4 (LPS) | ||||||||||
| ALL | 32 | 105 (2.2) | 72 | 108 (3.0) | 3 | (−31 to 51) | 0.90 | 5 | (−15 to 32) | 0.66 |
| Male | 16 | 100 (2.2) | 34 | 115 (2.5) | 14 | (−30 to 87) | 0.59 | 17 | (−9 to 54) | 0.22 |
| Female | 16 | 110 (2.3) | 38 | 103 (3.3) | −7 | (−50 to 66) | 0.81 | −7 | (−77 to 34) | 0.70 |
| M vs. F | 21 | (−38 to 144) | 0.59 | 24 | (−12 to 86) | 0.27 | ||||
| TLR5 (Flagellin) | ||||||||||
| ALL | 33 | 92 (2.5) | 69 | 112 (2.6) | 19 | (−17 to 72) | 0.34 | 7 | (−9 to 28) | 0.44 |
| Male | 17 | 105 (2.5) | 34 | 110 (3.2) | 5 | (−45 to 95) | 0.88 | 2 | (−21 to 38) | 0.88 |
| Female | 16 | 81 (2.5) | 35 | 113 (2.3) | 32 | (−13 to 107) | 0.20 | 11 | (−25 to 37) | 0.28 |
| M vs. F | −27 | (−87 to 104) | 0.47 | −9 | (−36 to 38) | 0.58 | ||||
| TLR7/8 (CLO75) | ||||||||||
| ALL | 32 | 95 (3.4) | 68 | 123 (3.5) | 28 | (−21 to 111) | 0.32 | 13 | (−11 to 47) | 0.32 |
| Male | 16 | 100 (3.4) | 34 | 112 (2.6) | 12 | (−44 to 120) | 0.75 | 28 | (−6 to 82) | 0.12 |
| Female | 16 | 89 (3.5) | 34 | 132 (4.3) | 42 | (−27 to 189) | 0.31 | −3 | (−78 to 44) | 0.89 |
| M vs. F | −31 | (−102 to 165) | 0.59 | 31 | (−13 to 113) | 0.22 | ||||
IL-12(p70) levels were analysed in culture supernatants from whole blood stimulated for 16 h with TLR ligands. The table shows geometric mean (geo mean) plus geometric standard deviation (GSD) cytokine levels in pg/mL. The mean difference (Δ; 95% confidence intervals; p-values) between those geometric means were estimated using general linear modelling (unadjusted and adjusted for covariates) with missing data substituted by multiple imputation. The difference between the effect of HCMV in males compared with females was estimated (M vs. F). N= number of infants analysed for each TLR ligand.
Table 6.
Effect of HCMV infection on TLR ligand-stimulated IL-10 responses by infant sex.
| IL-10 | HCMV− | HCMV+ | Comparison (Unadjusted) | Comparison (Adjusted) | ||||||
|---|---|---|---|---|---|---|---|---|---|---|
| N | Geo Mean (GSD) | N | Geo Mean (GSD) | Δ | 95%CI | p-Value | Δ | 95%CI | p-Value | |
| Unstimulated | ||||||||||
| ALL | 34 | 76 (8.0) | 73 | 96 (8.4) | 20 | (−35 to 148) | 0.59 | 136 | (−350 to 875) | 0.64 |
| Male | 17 | 149 (9.1) | 35 | 108 (9.2) | −40 | (−119 to 245) | 0.63 | 32 | (−596 to 1172) | 0.94 |
| Female | 17 | 39 (5.8) | 38 | 89 (8.1) | 49 | (−7 to 208) | 0.12 | 221 | (−1363 to 1368) | 0.58 |
| M vs. F | −90 | (−177 to 365) | 0.18 | −189 | (−988 to 1675) | 0.73 | ||||
| TLR2 (HKLM) | ||||||||||
| ALL | 35 | 4295 (3.3) | 73 | 3409 (4.3) | −886 | (−2258 to 1409) | 0.38 | 98 | (−243 to 563) | 0.62 |
| Male | 18 | 4786 (3.8) | 35 | 3366 (4.6) | −1420 | (−3273 to 2699) | 0.39 | −134 | (−530 to 437) | 0.60 |
| Female | 17 | 3830 (2.8) | 38 | 3441 (4.3) | −389 | (−2035 to 2768) | 0.75 | 283 | (−902 to 1120) | 0.33 |
| M vs. F | −1031 | (−3197 to 5038) | 0.64 | −418 | (−1024 to 728) | 0.27 | ||||
| TLR4 (LPS) | ||||||||||
| ALL | 32 | 3820 (3.7) | 72 | 3593 (4.0) | −227 | (−1744 to 2398) | 0.83 | 299 | (−348 to 1199) | 0.41 |
| Male | 16 | 5016 (2.5) | 34 | 3775 (3.6) | −1241 | (−3007 to 2078) | 0.38 | −533 | (−1260 to 553) | 0.29 |
| Female | 16 | 2910 (4.8) | 38 | 3475 (4.5) | 566 | (−1455 to 5393) | 0.69 | 860 | (−859 to 2297) | 0.062 |
| M vs. F | −1807 | (−4294 to 5484) | 0.40 | −1393 | (−2409 to −495) | 0.034 | ||||
| TLR5 (Flagellin) | ||||||||||
| ALL | 33 | 4409 (3.4) | 69 | 5412 (2.6) | 1003 | (−1040 to 4285) | 0.40 | 627 | (−226 to 1753) | 0.17 |
| Male | 17 | 4901 (3.4) | 34 | 5327 (2.5) | 425 | (−2125 to 5319) | 0.80 | 590 | (−637 to 2483) | 0.40 |
| Female | 16 | 3940 (3.6) | 35 | 5474 (2.8) | 1534 | (−1180 to 6917) | 0.35 | 661 | (−1569 to 2214) | 0.27 |
| M vs. F | −1109 | (−4366 to 7274) | 0.61 | −71 | (−1593 to 2630) | 0.94 | ||||
| TLR7/8 (CLO75) | ||||||||||
| ALL | 32 | 3941 (3.2) | 68 | 4198 (5.1) | 257 | (−1537 to 3391) | 0.82 | 350 | (−266 to 1148) | 0.29 |
| Male | 16 | 4382 (4.3) | 34 | 3786 (4.6) | −596 | (−2826 to 4825) | 0.75 | −580 | (−1416 to 684) | 0.32 |
| Female | 16 | 3544 (2.2) | 34 | 4534 (5.6) | 990 | (−1201 to 5231) | 0.47 | 1069 | (337 to 2195) | 0.005 |
| M vs. F | −1586 | (−4121 to 6083) | 0.49 | −1649 | (−2704 to −194) | 0.016 | ||||
IL-10 levels were analysed in culture supernatants from whole blood stimulated for 16 h with TLR ligands. The table shows geometric mean (geo mean) plus geometric standard deviation (GSD) cytokine levels in pg/mL. The mean difference (Δ; 95% confidence intervals; p-Values) between those geometric means were estimated using general linear modelling (unadjusted and adjusted for covariates) with missing data substituted by multiple imputation. The difference between the effect of HCMV in males compared with females was estimated (M vs. F). N = number of infants analysed for each TLR ligand. Bold are the significant effects.
Table 7.
Effect of HCMV infection TLR ligand-stimulated TNF-α:IL-10 ratio in culture supernatants by infant sex.
Table 7.
Effect of HCMV infection TLR ligand-stimulated TNF-α:IL-10 ratio in culture supernatants by infant sex.
| TNF:IL-10 | HCMV− | HCMV+ | Comparison (Unadjusted) | Comparison (Adjusted) | ||||||
|---|---|---|---|---|---|---|---|---|---|---|
| N | Geo Mean (GSD) | N | Geo Mean (GSD) | Δ | 95% CI | p-Value | Δ | 95% CI | p-Value | |
| Unstimulated | ||||||||||
| ALL | 34 | 0.95 (3.0) | 73 | 0.97 (3.0) | 0.02 | (−0.33 to 0.56) | 0.94 | 0.18 | (−0.08 to 0.54) | 0.19 |
| Male | 17 | 0.93 (2.8) | 35 | 1.03 (2.7) | 0.10 | (−0.36 to 0.92) | 0.75 | 0.33 | (−0.02 to 0.86) | 0.064 |
| Female | 17 | 0.97 (3.3) | 38 | 0.93 (3.3) | −0.04 | (−0.49 to 0.82) | 0.89 | 0.04 | (−1.08 to 0.60) | 0.86 |
| M vs. F | 0.14 | (−0.46 to 1.59) | 0.75 | 0.29 | (−0.18 to 1.18) | 0.26 | ||||
| TLR2 (HKLM) | ||||||||||
| ALL | 35 | 3.62 (2.8) | 73 | 2.57 (3.1) | −1.04 | (−1.93 to 0.32) | 0.12 | −0.74 | (−1.29 to 0.05) | 0.063 |
| Male | 18 | 2.52 (2.5) | 35 | 2.33 (2.5) | −0.19 | (−1.13 to 1.39) | 0.77 | −0.18 | (−0.90 to 1.04) | 0.72 |
| Female | 17 | 5.30 (2.8) | 38 | 2.77 (3.6) | −2.53 | (−3.80 to −0.19) | 0.038 | −1.48 | (−4.07 to −0.27) | 0.021 |
| M vs. F | 2.34 | (−1.06 to 5.20) | 0.16 | 1.30 | (−0.38 to 3.17) | 0.20 | ||||
| TLR4 (LPS) | ||||||||||
| ALL | 32 | 1.66 (3.3) | 72 | 1.29 (3.4) | −0.37 | (−0.88 to 0.46) | 0.32 | −0.09 | (−0.58 to 0.66) | 0.77 |
| Male | 16 | 1.22 (3.0) | 34 | 1.33 (3.0) | 0.11 | (−0.53 to 1.35) | 0.79 | 0.03 | (−0.58 to 1.17) | 0.95 |
| Female | 16 | 2.27 (3.4) | 38 | 1.26 (3.8) | −1.00 | (−1.64 to 0.29) | 0.10 | −0.22 | (−2.03 to 0.95) | 0.64 |
| M vs. F | 1.12 | (−0.29 to 3.27) | 0.17 | 0.25 | (−0.52 to 2.07) | 0.70 | ||||
| TLR5 (Flagellin) | ||||||||||
| ALL | 33 | 1.19 (3.3) | 69 | 0.82 (3.9) | −0.37 | (−0.70 to 0.18) | 0.16 | −0.12 | (−0.50 to 0.47) | 0.63 |
| Male | 17 | 1.05 (4.2) | 34 | 0.81 (3.7) | −0.24 | (−0.69 to 0.78) | 0.54 | 0.08 | (−0.39 to 0.98) | 0.79 |
| Female | 16 | 1.36 (2.5) | 35 | 0.83 (4.1) | −0.53 | (−0.91 to 0.16) | 0.11 | −0.36 | (−1.74 to 0.50) | 0.34 |
| M vs. F | 0.30 | (−0.22 to 1.73) | 0.64 | 0.44 | (−0.14 to 1.78) | 0.38 | ||||
| TLR7/8 (CLO75) | ||||||||||
| ALL | 32 | 1.00 (2.7) | 68 | 0.84 (3.2) | −0.16 | (−0.46 to 0.30) | 0.43 | −0.06 | (−0.42 to 0.45) | 0.78 |
| Male | 16 | 0.78 (2.9) | 34 | 0.71 (2.8) | −0.07 | (−0.40 to 0.55) | 0.76 | −0.09 | (−0.48 to 0.60) | 0.75 |
| Female | 16 | 1.29 (2.4) | 34 | 0.96 (3.5) | −0.33 | (−0.75 to 0.41) | 0.31 | −0.02 | (−1.24 to 0.92) | 0.95 |
| M vs. F | 0.26 | (−0.15 to 1.20) | 0.65 | −0.06 | (−0.58 to 1.05) | 0.86 | ||||
TNF:IL-10 ratios were analysed in culture supernatants from whole blood stimulated for 16 h with TLR ligands. The table shows geometric mean (geo mean) plus geometric standard deviation (GSD) cytokine levels in pg/m. The mean difference (Δ; 95% confidence intervals; p-values) between those geometric means were estimated using general linear modelling (unadjusted and adjusted for covariates) with missing data substituted by multiple imputation. The difference between the effect of HCMV in males compared with females was estimated (M vs. F). N = number of infants analysed for each TLR ligand. Bold are the significant effects.
© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
Share and Cite
MDPI and ACS Style
Cox, M.; Adetifa, J.U.; Noho-Konteh, F.; Sanyang, L.C.; Drammeh, A.; Plebanski, M.; Whittle, H.C.; Rowland-Jones, S.L.; Robertson, I.; Flanagan, K.L. Sex-Differential Impact of Human Cytomegalovirus Infection on In Vitro Reactivity to Toll-Like Receptor 2, 4 and 7/8 Stimulation in Gambian Infants. Vaccines 2020, 8, 407. https://doi.org/10.3390/vaccines8030407
AMA Style
Cox M, Adetifa JU, Noho-Konteh F, Sanyang LC, Drammeh A, Plebanski M, Whittle HC, Rowland-Jones SL, Robertson I, Flanagan KL. Sex-Differential Impact of Human Cytomegalovirus Infection on In Vitro Reactivity to Toll-Like Receptor 2, 4 and 7/8 Stimulation in Gambian Infants. Vaccines. 2020; 8(3):407. https://doi.org/10.3390/vaccines8030407
Chicago/Turabian StyleCox, Momodou, Jane U. Adetifa, Fatou Noho-Konteh, Lady C. Sanyang, Abdoulie Drammeh, Magdalena Plebanski, Hilton C. Whittle, Sarah L. Rowland-Jones, Iain Robertson, and Katie L. Flanagan. 2020. "Sex-Differential Impact of Human Cytomegalovirus Infection on In Vitro Reactivity to Toll-Like Receptor 2, 4 and 7/8 Stimulation in Gambian Infants" Vaccines 8, no. 3: 407. https://doi.org/10.3390/vaccines8030407
APA StyleCox, M., Adetifa, J. U., Noho-Konteh, F., Sanyang, L. C., Drammeh, A., Plebanski, M., Whittle, H. C., Rowland-Jones, S. L., Robertson, I., & Flanagan, K. L. (2020). Sex-Differential Impact of Human Cytomegalovirus Infection on In Vitro Reactivity to Toll-Like Receptor 2, 4 and 7/8 Stimulation in Gambian Infants. Vaccines, 8(3), 407. https://doi.org/10.3390/vaccines8030407
Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.
