Systemic and Mucosal Concentrations of Nine Cytokines Among Individuals with Neisseria gonorrhoeae infection in Nairobi Kenya

Introduction: The human-restricted sexually transmitted Neisseria gonorrhoeae (NG) has been shown to modulate the immune response against it and consequently the cytokines produced. The levels of cytokines in NG infection in the African population have not been well described. We aimed to quantify the systemic and mucosal cytokines in NG infection. Methods: This was a comparative cross-sectional study. Levels of nine cytokines (IL-1β, IL-2, IL-4, 1L-6, 1L-10, 1L-12p70, IL-17A, TNF-α and INF-γ) were measured from plasma and genital samples (urethral swabs in men and cervicovaginal lavage in women) from 61 Neisseria gonorrhoeae infected individuals seeking treatment for sexually transmitted infections (STIs) at Casino Health Centre in Nairobi, Kenya. A comparative group of 61 NG-uninfected individuals, seeking treatment at the same facility but with laboratory-confirmed negative Neisseria gonorrhoeae, Chlamydia trachomatis (CT), Mycoplasma genitalium (MG) and Trichomonas vaginalis(TV) was also included. The Mann-Whitney U test was used to compare the cytokine levels between NG-infected and uninfected individuals. Data was analyzed using STATA ver. 15.1. Results: Overall, systemic IL-6, TNF-α and IL-10 were elevated while genital IL-10 and TNF-α were lower in NG positive participants. On subgroup analysis by sex, the levels of genital IL-1β and IL-6 and systemic IL-6 were elevated in NG-infected men. None of the genital cytokines were elevated in NG-infected women, while all systemic cytokines, except INF-γ, were elevated in NG-infected women. Conclusions: Neisseria gonorrhoeae induced the production of different cytokines in men and women, with men having a pro-inflammatory genital response. These differences should be taken into consideration during development of various interventions e.g. vaccine development.


Introduction
Neisseria gonorrhoeae (NG) is a strict human pathogen 1,2 .This monoxenous cycle has resulted in the bacterium's ability to evade both innate and adaptive immune responses mounted against it.Despite the fact that the immune system mounts a robust inflammatory response against NG, immunological memory that would lead to protective immunity against uncomplicated infections has not been defined 3 .To overcome the innate immune responses, it is able to prevent phagocytosis by neutrophils 4,5 , or, if phagocytosis occurs, to survive inside the phagocytic cells 5 .Further, it is able to evade the host's nutritional immunity which ensures its survival in the host 6 .Not only is it able to evade the immune response but also to modulate it.Modulation of the immune response may involve manipulating the response to that which it is able to overcome or evade 7 , influencing the subtype of immune response cells produced 8 or modulation of cell death pathways 9 .To modulate the immune response, NG drives the differentiation of naïve CD4 + T helper (Th) cells to the Th17 axis while actively suppressing Th1 and Th2 responses 7 .It also induces the production of interleukin 10 (IL-10).IL-10 suppresses the exacerbated inflammatory response.Eventually, the adaptive immune response may also be suppressed 10 .Consequently, there is increased production of pro-inflammatory cytokines e.g.IL-1β, IL-6, TNF-α, IL-17A.The result is an influx of neutrophils to the site of infection in symptomatic cases but with no immunological memory 10 .This ability of NG to evade and modulate the protective response from its human host is happening against a backdrop of increasing reports of antimicrobial resistant (AMR) isolates from around the world [11][12][13][14] .The current recommended treatment for NG is an extended spectrum cephalosporin (cefixime or ceftriaxone) in combination with azithromycin 15,16 .However, cases of both multi-drug resistant (MDR) and extensively drug resistant (XDR) strains have been reported and continue to be reported around the world 17 .New strategies to combat NG infection are therefore urgently needed.Consequently, NG has been placed in the global high priority pathogen list requiring research and development efforts for new antimicrobial agents 18 .In addition, the WHO, has set a target of 90% reduction in incident cases of NG by 2030 19 , which, although has low mortality, causes significant complications.
The complications of NG infection are of great public health concern.Some of the complications in women include pelvic inflammatory disease (PID) which is a chronic debilitating infection; ectopic pregnancy, rupture of which causes haemorrhage which is the leading cause of maternal mortality; and tubal factor infertility which is one of the commonest causes of infertility 20 .Complications in men include infertility while in neonates it causes ophthalmia neonatorum 21 which can lead to blindness if not treated early.In addition, NG has been associated with the risk of acquisition of other infections such as HIV-1 22 .Other potentially fatal conditions which have been described include endocarditis and sepsis 23,24 .
Strategies that can be used to combat antimicrobial resistant pathogens include vaccination 25 and use of new therapeutics.
However the search for an NG vaccine has so far been disappointing 26 .Knowledge of the cytokine profile in NG infection in different populations will be useful in informing future strategies.The purpose of this study was to determine the systemic and mucosal concentrations of nine cytokines among individuals seeking treatment for sexually transmitted infections in Nairobi, Kenya.

Study setting
Between April and June 2019, we conducted a cross-sectional comparative study of consecutive adults, 18 to 49 years, seeking treatment for sexually transmitted infections at Casino Special Treatment Centre (STC) Health Centre in Nairobi, Kenya.Patients were included if they were between 18 and 49 years and provided written informed consent to participate in the study.Pregnant women were excluded from the study because of the invasive nature of specimen collection (use of specula).Menstruating women were also excluded because the protein-rich menstrual blood has been shown to interfere with mucosal cytokine levels 27 .

Sociodemographic data collection
An interviewer-administered questionnaire was used to collect the sociodemographic characteristics and the sexual history.It assessed age, marital status, level of education, occupation, monthly income, circumcision status in men, menarche, sexual orientation, sexual partners in the preceding one year and new sexual partners in the preceding three months.The reported symptoms assessed were discharge and dysuria in men; and discharge, dysuria, vaginal itch, intermenstrual bleeding, post-coital bleeding and lower abdominal pain (LAP) in women.

Genital sample collection
Vaginal samples were collected after insertion of a speculum.For detection of Neisseria gonorrhoeae infection, a dacron swab was placed approximately 2cm into the os and rotated three times, removed and placed into an empty tube and immediately placed on ice.Cervicovaginal lavage (CVL) was collected for mucosal cytokines.Briefly, the cervix and the lateral vaginal walls were flushed with 10 ml normal saline at room temperature.The fluid was allowed to pool into the posterior fornix and aspirated into the same syringe.This procedure was repeated 3-5 times with the same fluid.The fluid was then collected and placed in a 15ml falcon tube and immediately placed on ice to await transportation, up to four hours.Urethral samples were collected by gently inserting dacron swabs 2-4 cm inside the urethra and rotating three times.They were then carefully removed and placed (i) into an empty tube for detection of Neisseria gonorrhoeae infection and (ii) in a tube containing 1.5ml phosphate buffered saline (PBS) for mucosal cytokine analysis.The tubes were immediately placed on ice to await transportation.
For systemic cytokine analysis, 10mls of venous blood was collected, from all participants, into EDTA treated tubes and then immediately placed on ice to await transportation.
All the samples were transported within four hours to the Department of Medical Microbiology, University of Nairobi.Once in the laboratory, the blood was centrifuged at 2000 g for 10 minutes at 4°C After centrifugation, the vacutainers were carefully removed from the centrifuge making sure that the tubes were not inverted, to avoid remixing of the plasma with the cells.Using a transfer pipette, the supernatant was carefully aliquoted into 2ml cryovials and immediately stored at -70°C to await analysis.Cells within the cervicovaginal lavage fluid were separated by centrifugation at 1000 rpm for 10 minutes at 4°C.The supernatant was then stored in 2 ml aliquots at -70°C to await analysis.

Screening for HIV and STIs
All participants were tested for HIV using Determine HIV Rapid Test (Abbott Diagnostics) with confirmatory testing being done with the First Response™ (Premier Medical Corporation Private Ltd., Gujarat, India), according to Kenya's Ministry of Heath guidelines.Those who tested negative with the first test were considered HIV negative while those who tested positive with the confirmatory test were considered HIV positive and were either enrolled in the Casino H/C HIV comprehensive care clinic (CCC) or referred to a health facility of their choice for follow-up.
Screening for Neisseria gonorrhoeae, Chlamydia trachomatis, Trichomonas vaginalis and Mycoplasma genitalium was done using Multiplex PCR (Sacace Biotechnologies,Como, Italy) according to the manufacturer's instructions.

Measurement of cytokines
The systemic and genital levels of 9 cytokines (IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12p70, IL-17A, TNF-α and IFN-γ) were measured using the Invitrogen™ High Sensitivity 9-Plex Human ProcartaPlex™ Panel (Thermo Fisher Scientific Inc.) Briefly, samples were first thawed.Fifty μL of diluted magnetic beads were added to each well.The plates were washed and samples and standards added to the wells (25 μL of samples and 25 μL of standards).The plates were then shaken at room temperature for 30 min, after which they were transferred at 4°C in the dark.Shaking for 30 minutes at room temperature was then done and 25 μL of Detection Antibody Mix was added followed by incubation with shaking at room temperature for 30 min in the dark.The beads were then washed twice, 50 μL of Streptavidin-PE was added and the beads washed twice again.Incubation with shaking at room temperature for 30 min in the dark was done followed by washing twice.50 μL of Amplification Reagent 1 and 2 were added with shaking at room temperature for 30 min in the dark.50 μL of Amplification Reagent 2 was added followed by incubation with shaking at room temperature for 30 min in the dark.The beads were then washed twice.Reading buffer (120 μL) was added and shaking at room temperature for 5 min in the dark done.Data was acquired on the Magpix™ system.

Statistical analysis
Data was entered into RedCap 28 hosted at APHRC and analysed using STATA version 15.1.Categorical variables were compared using the χ2 or Fisher's exact test.Cytokine levels were compared using the Mann-Whitney U test or the Student t test after tests of normality using the Shapiro-Wilk test.The level of statistical significant was set at <0.05.

Ethical approval
The study received ethical approval from the Kenyatta National Hospital/University of Nairobi (KNH/UoN) ERC (Protocol number (P304/06/2017).Permission to conduct the study at Casino H/C was granted by Nairobi County's Department of Health Services.

Demographic characteristics
Between April and June 2019, a total of 297 individuals, 18 to 49 years, were recruited.Of these, 122 were included in this comparative cross-sectional study (61 NG positive, and 61 NG negative).Co-infections with three organisms: Chlamydia trachomatis(CT), Mycoplasma genitalium(MG) and Trichomonas vaginalis(TV) were excluded from those in the NG infection group; while infections with four organisms: NG, CT, TV and MG were excluded from those in the NG negative group.MS Excel random number generator was used to select participants in the NG negative group.
Table 1 summarizes the sociodemographic characteristics, reported symptoms at recruitment and sexual history of the study participants.There was a significant difference between infected and uninfected participants with regards to sex composition (p=<0.001).In addition, there was a significant difference between groups in participants reporting dysuria, number of sexual partners and having a new sexual partner in the previous one year.

Concentration of cytokines
Table 2 shows the concentration of cytokines in the two groups.In the systemic cytokines, more than half of the study participants in both groups had undetectable levels of IL-10 and TNF-α.The NG positive participants had higher concentrations of IL-6, IL-10 and TNF-α (p= 0.028, 0.052 and 0.012 respectively).
In the genital cytokines, more than half of the participants had undetectable levels of IL-2, IL12p70, and IFN-γ.Further, only 4 participants had detectable IL-4 levels.The levels of IL-10 and TNF-α were statistically significantly lower in NG positive individuals than in NG negative individuals (both p=0.001)

Subgroup analysis of cytokine concentration by sex
We proceeded to carry out subgroup analysis of cytokine concentration by sex since the sex composition was statistically significantly different in both groups (p=<0.001,Table 1).Table 3 shows the subgroup analysis of cytokine concentrations by sex.In female participants, systemic IL-10 and IFN-γ were detectable in less than half of the participants.Genital IL-4 and IL-12p70 were not detectable in NG positive participants.
All systemic cytokines except IFN-γ were present in higher concentrations in NG infected women than in NG negative women.However, there were no statistically significant differences in the genital cytokine concentrations in the nine analysed cytokines (Table 3).
In male participants, systemic IL-10 and TNF-α were undetectable in more than half of the NG positive participants.Genital IL-4 was detectable in only two participants.On the other hand, systemic IL-1β was detectable in all the participants.
In NG negative male participants, systemic IL-1β was detectable in all participants while only one participant had detectable levels of IL-10.In genital cytokines, only IL-1β was detectable in more than half of the participants with other cytokines being detected in none or only a few participants : ranging from 0/21; 0% (IL-4) to 6/21; 28.67%(IL-12p70 and IFN-γ).
The levels of systemic and genital IL-6 and genital IL-1β were statistically significantly higher in NG positive men compared to NG negative men (p=0.010,0.038 and <0.001 respectively).There were no significant differences in the other cytokines (Table 3).

Discussion
In this study, we assessed the genital and systemic levels of nine cytokines: IL-1β, IL-2, IL-4 IL-6, IL-10, IL-12p70, IL-17A, IFN-γ and TNF-α in Neisseria gonorrhoeae infected individuals.Overall, the levels of systemic IL-6, TNF-α and IL-10 were elevated while genital IL-10 and TNF-α were lower in NG positive participants.By contrast, some studies have shown that NG induces an anti-inflammatory state with increased production of IL-10 in the genital tract 10,29 while another study showed increased TNF-α production leading to protection of NG-infected cells from undergoing apoptosis 30 .However, with evidence showing that NG uses different mechanisms to establish infection in the male and female genital tracts 31 , and that the immune response is likely to be gender specific, the cytokine profile in different gender should be considered differently.
In our study, NG-infected men had elevated genital IL-1β and IL-6 as well as elevated systemic IL-6.This is similar to findings by Ramsey et al. (1995) of sequential increase in IL-6 in urine before the onset of symptoms and of IL-1β at the onset of symptoms in some of the participants 32 .Expression of genital IL-1β has been associated with inflammation and symptomatic infection and was significant in disease progression 33 .IL-6 production is important in clearance of infection and restoration of damaged tissue 34 .
In women, none of the assayed genital cytokines was elevated.This is similar to findings by Hedges et al. (1998) and Cauci & Culhane (2007), showing that NG had no effect on genital cytokines 35,36 .In contrast, NG was associated with elevated genital cytokines (IL-1β, IL-12p70, TNF-α, IL-2, and IL-17 among others) in a study involving HIV-infected and uninfected women in South Africa 37,38 .The lack of upregulation of any of the assayed cytokines could be explained by the fact that NG has been shown to modulate the immune response and suppress proinflammatory responses 39 .On the other hand, eight out of the nine assayed systemic cytokines were elevated in NG-positive women.
Immune responses at the mucosal level have been associated with systemic responses.Indeed, Morrison et al. (2020) argue that the imbalances of systemic and genital cytokines may explain the increased risk of acquisition of some infections e.g.HIV 40 and therefore stressing the need of considering both responses at the mucosal and the systemic level.
Our study had several limitations.First, we did not screen for bacterial vaginosis (BV).BV has been associated with a pro-inflammatory response in the genital tract and is prevalent in the African population 41 .Secondly, we did not quantify the hormonal levels in women.Progesterone has been associated with inhibition of inflammatory effects of NG infection and a reduction of genital levels of IL-1β, TNF-α, and IL-6 with 42 resultant asymptomatic infection.Future studies should explore the relationship between cytokine and progesterone levels.Thirdly, the microenvironments evaluated are quite heterogenous.This has the potential to interfere with the significance and interpretation of the results obtained.Lastly, we used the lower limits of detection set by the manufacturer.Given that polypeptides are synthesized and secreted at different intensities depending on the microenvironment evaluated, there is the potential for experimental bias.Our results can therefore not be generalized to the general population.
Despite the limitations, our study quantifies nine cytokine profile in NG and shows differences in men and women.Future studies can explore these differences using a larger sample size and controlling for possible confounders such as progesterone levels and bacterial vaginosis.

Data availability
Mendeley Data.Systemic and mucosal concentrations of nine cytokines among individuals with Neisseria gonorrhoeae infection in Nairobi, Kenya.DOI: 10.17632/67b55dncm7.1 43 This project contains the following underlying data: -We conducted a cross-sectional comparative study of consecutive adults, 18 to 49 years, seeking treatment for sexually transmitted infections at Casino Special Treatment Centre (STC) Health Centre in Nairobi, Kenya.
-Patients were included if they were between 18 and 49 years and provided written informed consent to participate in the study.An interviewer-administered questionnaire was used to collect the sociodemographic characteristics and the sexual history.Vaginal samples were collected after insertion of a speculum.For detection of Neisseria gonorrhoeae infection, a dacron swab was placed approximately 2cm into the os and rotated three times, removed and placed into an empty tube and immediately placed on ice.Cervicovaginal lavage (CVL) was collected for mucosal cytokines.
-Briefly, the cervix and the lateral vaginal walls were flushed with 10 ml normal saline at room temperature.
The fluid was allowed to pool into the posterior fornix and aspirated into the same syringe.This procedure was repeated 3-5 times with the same fluid.
-The fluid was then collected and placed in a 15ml falcon tube and immediately placed on ice to await transportation, up to 4 hours.Urethral samples were collected by gently inserting dacron swabs 2-4 cm inside the urethra and rotating three times.They were then carefully removed and placed (i) into an empty tube for detection of Neisseria gonorrhoeae infection and (ii) in a tube containing 1.5ml phosphate buffered saline (PBS) for mucosal cytokine analysis.The tubes were immediately placed on ice to await transportation.
-For systemic cytokine analysis, 10mls of venous blood was collected, from all participants, into EDTA treated tubes and then immediately placed on ice to await transportation.All participants were tested for HIV using Determine HIV Rapid Test (Abbott Diagnostics) with confirmatory testing being done with the First Response™ (Premier Medical Corporation Private Ltd., Gujarat, India), according to Kenya's Ministry of Heath guidelines.
-Those who tested negative with the first test were considered HIV negative while those who tested positive with the confirmatory test were considered HIV positive and were either enrolled in the Casino H/C HIV comprehensive care clinic (CCC) or referred to a health facility of their choice for follow-up.Screening for Neisseria gonorrhoeae, Chlamydia trachomatis, Trichomonas vaginalis and Mycoplasma genitalium was done using Multiplex PCR (Sacace Biotechnologies, Como, Italy) according to the manufacturer's instructions.
Have the authors examined if these individuals are also free of other types of infection?Or is it because the detection limit is very low using this highly sensitive kit?It would be good if a dot plot can be provided.

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility?Yes

Are the conclusions drawn adequately supported by the results? Partly
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Immunology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
patients with NG, CT, TV, and MG co-infections were excluded from further analysis but not HIV (why so?).I would have loved to see the effect of these co-infections on the cytokine profiles if they indeed had no effect then exclusion would not have been important and this would have increased the power of the study.
The data analysis section needs to be expanded as it's not clear why the authors used nonparametric tests.Did the authors just assume that the data was not normally distributed, otherwise, an analysis of normality for the data set should have been done to guide downstream analyses?It would also be helpful to indicate which tests were done for which data set.
○ Furthermore, I do not buy the argument of comparing genital cytokine levels between females and males as indicated in table 3. Mucosal NG in both sexes presents differently and such a comparison would not yield.Indeed, even the sample collection between the 2 was from different anatomical structures making comparisons impossible.I would advise to delete this from the table 3 and concentrate on systemic cytokines.

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My thinking is also that this study would have provided more insights into the different clinical spectrum for NG.I would therefore have loved to see how the cytokine profiles would vary across the different clinical presentations as this would have made the work more novel and interesting.For example, in this study, did all males and females present with the same clinical spectrum, and if not do cytokines have role to play?Or are individuals with less severe clinical spectrum associated with a given cytokine profile?

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The discussion section of the paper, needs to be well articulated with more evidence.The implication of elevated systemic cytokines in NG patients needs to be elucidated and what this would mean on disease prognosis.

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The cytokine nomenclature is also not consistent and needs to be clarified.In some cases, IFN-g or TNF-a or b are used.Standard nomenclature for these should be used throughout and consistently throughout the manuscript.

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For all equipment mentioned, the manufacturer and country of origin should be indicated.Reviewer Expertise: Clinical Immunology and population genetics I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

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Is the work clearly and accurately presented and does it cite the current literature?PartlyIs the study design appropriate and is the work technically sound?YesAre sufficient details of methods and analysis provided to allow replication by others?PartlyIf applicable, is the statistical analysis and its interpretation appropriate?PartlyAre all the source data underlying the results available to ensure full reproducibility?PartlyAre the conclusions drawn adequately supported by the results?PartlyCompeting Interests: No competing interests were disclosed.

Table 1 . Sociodemographic characteristics of the study participants.
± Female participants only † Male participants only