Influence of H. Pylori and Sex on Leukocyte Differentials


 Background: Influence of Helicobacter pylori infection and sex difference on leukocyte differentials was insufficiently understood. We therefore conducted the current study to evaluate influence of H. pylori and sex on peripheral leukocyte differentials as well as influence of Helicobacter pylori eradication. Methods: Dyspeptic patients and persons asked to examine gastroduodenal lesions at medical check were included. Total leukocytes, differentials, and anti-H.pylori IgG antibody were measured. Those change after eradication was also measured. H. pylori infection was assessed by anti-H. pylori IgG antibody, rapid urease test, and pathologic findings. H. pylori-uninfected patients and non-dyspeptic/seronegative persons were regarded as negative controls. Results: Totally 374 patients and 299 controls were evaluated, and 167 patients were successfully eradicated. Peripheral counts of neutrophil and monocyte elevated with H. pylori infection: 3221.7+/-1108.7 vs. 2911.9+/-1027.3/μL and 307.5+/-130.5 vs. 281.5+/-106.4/μL, (p­=.0002 and .0054). Compared to females, males manifested elevated counts of every leukocyte, but the difference was insignificant in basophil (p=.0089, 0.0316, <.0001, and .0384 for neutrophil, lymphocyte, monocyte, and eosinophil, respectively). After eradication, counts of neutrophil, lymphocyte, and monocyte declined: 3111.0+/-966.8 to 2785.1+/997.2/μL, 1905.9+/-603.2 to 1831.6+/-613.5/μL, and 293.1+/-113.3 to 264.3+/-93.6/μL by 2 months (p=<.0001, .0189, and .0004). In contrast, eosinophil counts elevated from 123.2+/-97.0 to 139.8+/-115.4 by 2 months, and to 159.6+/-132.8/μL by 6 months (p=.0349 and <.0001). Conclusions: We confirmed increases in neutrophils and monocytes in H. pylori-infected patients. Successful eradication reduced peripheral counts of neutrophil, lymphocyte, and monocyte, whereas it increased eosinophil counts. Males manifested elevated counts of every leukocyte, comparing to females. H. pylori infection influences systemic immune response and may not predispose to allergic disorders.Trial registration: The study protocol was registered on UMIN (University hospital Medical Information Network system) in Japan (R000017345) in 2014.


Background
Helicobacter pylori (H. pylori) infection had been reported to alter leukocyte differentials [1][2][3][4][5][6], and most of researchers reported increased numbers of total leukocytes, neutrophil, and monocyte, comparing to those uninfected. Concerning eosinophil and basophil, no speci c alterations were reported. In addition, most of researchers did not pay attention to some factors that could in uence the leukocyte differentials such as smoking, medications, sex difference, and additional morbidity. We therefore conducted the current study to elucidate in uence of H. pylori infection and sex difference on leukocyte differentials in detail.

Methods
Dyspeptic patients and persons recommended to receive close examination at medical check were included in the current study. After obtaining informed/written consents to check immunohematologic parameters and H. pylori infection, they were venipunctured to examine blood tests including complete blood counts, leukocyte differentials, and anti-H pylori IgG antibody. Because H pylori eradication therapy applies only to endoscopically diagnosed gastritis in Japan, patients received endoscopic examination when they wanted to eradicate H pylori. Diagnosis of gastritis was made according to the Kyoto classi cation of gastritis and that of gastroduodenal ulcer did according to the classi cation of Sakita and Miwa [7,8]. To evaluate H. pylori status, biopsy specimens were obtained from the antrum and body in the greater curvature of the stomach. The samples were placed into rapid urease test (RUT) kits, and the results were evaluated 5 hrs. later. These samples were also prepared for pathologic evaluation. Anti-H. pylori IgG antibody more than 10 U/ml was accepted as seropositive. We de ned H. pylori-infected patients to be positive for anti-H. pylori IgG antibody as well as for RUT or pathologic ndings of gastromucosal biopsy. The difference in total leukocytes counts and differentials were evaluated according to the presence or absence of H. pylori infection. We also evaluated the in uence of sex difference on the parameters. Moreover, some of H. pylori-infected patients received triple therapy mainly consisted of amoxicillin 1500 mg/day, clarithromycin 400 mg/day, and proton-pump inhibitor for a week. The change in the parameters after successful eradication was evaluate at 2, 6, and 12 months after the treatment.

Controls
Patients unmatched to have H. pylori infection and non-dyspeptic persons con rmed as seronegative for H. pylori were regarded as H. pylori-uninfected controls.

Patient exclusion
Patients with immunohematologic, rheumatic, malignant, peptic ulcer, and infectious diseases any other than H. pylori infection such as pneumonia, tuberculosis, hepatitis etc. were excluded. In addition, persons aged < 15yrs and those with prior eradication were excluded. Current smokers and ex-smokers who quitted smoking within 5 or less years were excluded [9,10]. Because non-steroidal antiin ammatory drugs (NSAIDs) might induce neutropenia and tend to bleed, NSAID users were also excluded. To further evaluate eosinophil counts, allergic patients were excluded when required.

Statistics
Data were expressed as mean+/-SD. Statistical difference was evaluated using Student's paired and unpaired t-tests, and p values less than .05 were accepted as signi cantly different.

Ethics and trial registration
The study protocol was registered on UMIN (University hospital Medical Information Network system) in Japan (R000017345). The institutional ethical committee of the Itabashi Hospital, Nihon University School of Medicine, approved the study protocol (RK141114-3, 2014.12.29).   (Table 2a)

Discussion
We here showed H. pylori infection elevates peripheral counts of both neutrophil and monocyte, and it elevates lymphocyte counts in females. We also showed males de nitely manifest elevated counts of monocyte, irrespective of H. pylori infection. Likewise, H. pylori-uninfected males showed elevated counts of eosinophil and lymphocyte, comparing to uninfected females. In addition, we disclosed successful H. pylori eradication cancelled such elevations in peripheral counts of neutrophil, lymphocyte, and monocyte, whereas it elevated peripheral eosinophil counts. The infection seemed to down-regulate peripheral eosinophil counts in males. Recently, some researchers paid attention to neutrophil/lymphocyte (N/L) ratio in investigating organ damage: they reported elevation of N/L ratio in H. pylori-infected patients, compared to uninfected controls [3][4][5][6]. Among them, Jafarzadeh A et al. reported elevated neutrophil counts in H. pylori-infected patients with gastric ulcer, compared to those of asymptomatic H. pylori-infected controls [3]. Sahin Y et al. reported elevated neutrophil counts and the decline after successful eradication among children [4]. Farah R et al. and Zeren S et al. reported elevated neutrophil counts and N/L ratio in H. pylori-infected patients [5,6]. The both researchers also reported lowered lymphocyte counts in H. pylori-infected patients, although they did not assess sex difference in the counts. In contrast, we disclosed elevated lymphocyte counts in H. pylori-infected females while we observed insigni cant reduction of lymphocyte counts in H. pylori-infected males. These 6 studies and the current data showed increases in peripheral neutrophils in H. pylori-infected patients. In addition, Hansen PS et al. had reported proin ammatory activation of neutrophils and monocytes in H. pylori-infected patients [11]. Taking these 7 studies and the current study into account, the increase in peripheral neutrophil counts could be a hull mark of H. pylori infection, and the increase in monocyte counts appears characteristic phenomenon in this infection.
We rstly present H. pylori infection could down-regulate peripheral eosinophil counts and its eradication de nitely elevated peripheral eosinophil counts, in the rst six months with a subsequent decline. H. pylori infection have been increasingly reported to inversely associate with allergic disorders probably due to the induction of regulatory T cells (T-regs) and skewed property to type 1 helper T cells (Th1) [12][13][14][15][16][17][18][19][20][21][22], although some of researchers expressed opposite opinion [23]. Our data could support such anti-allergic effect of the infection. When we accepted the increase in peripheral eosinophils as a predisposed parameter to allergic disorders, the current observations of both the decrease in eosinophils in H. pyloriinfected patients and the increase after successful eradication could indicate anti-allergic effect of the organism. The current data at least means H. pylori infection cannot predispose to allergic disorders. Likewise, some researchers reported aberration of eosinophil after successful eradication of H. pylori [24,25].
The reports assessing sex difference in leukocyte differentials are lacking, and few researchers paid attention to smoking status. Male sex has been reported to up-regulate peripheral monocyte counts [26][27][28], and Nah E et al. solely reported higher eosinophil counts in males than in females [26]. Taking their observations and the current data, the elevated monocyte counts in males over females seems de nitely and uniquely derived from male-sex. The current data further indicates males have signi cantly elevated counts of another sub-group of leukocytes, comparing to females in Japanese.

Conclusion
We here con rmed elevated counts of neutrophil and monocyte in H. pylori-infected patients. Successful eradication reduced peripheral counts of neutrophil, lymphocyte, and monocyte, whereas it increased eosinophil counts. H. pylori infection in uences systemic immune response and may not predispose to allergic disorders. In addition, males manifested de nite elevation of monocyte counts, comparing to females. Without in uence of H. pylori infection, males showed elevation of every sub-group of leukocytes in the peripheral blood, although the difference in neutrophil and basophil was insigni cant.

Limitations Of The Current Study
First, we evaluated the patients aged near 60-year-old in the current study. We therefore may not apply the current data for the youth. Second, due to a large number of excluded males such as smokers and ulcer patients, we were forced to evaluate a smaller number of male participants: male vs. female 219:438. Therefore, the difference derived from males could be underestimated.

Declarations
We performed the study according to our ethical standards as follows. After obtaining informed/written consents to check immunohematologic parameters and H. pylori infection, they were venipunctured to examine blood tests including complete blood counts, leukocyte differentials, and anti-H pylori IgG antibody. The personal information of the participants is kept strictly con dential.The study protocol was registered on UMIN (University hospital Medical Information Network system) in Japan (R000017345).

Funding
This research did not receive any speci c grant from funding agencies in the public, commercial, or notfor-pro t sectors.

Availability of data and materials
The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

Compliance with Ethical Standards
We performed the study according to our ethical standards as follows.
After obtaining informed/written consents to check immunohematologic parameters and H. pylori infection, they were venipunctured to examine blood tests including complete blood counts, leukocyte differentials, and anti-H pylori IgG antibody.
The personal information of the participants is kept strictly con dential.
The study protocol was registered on UMIN (University hospital Medical Information Network system) in Japan (R000017345). The institutional ethical committee of the Itabashi Hospital, Nihon University School of Medicine, approved the study protocol (RK141114-3, 2014.12.29) Consent for publication Not applicable.

Competing interests
The authors declare that they have no competing interest.
Disclosure of potential con icts with interest YM received honoraria for the lectures from Astra-zeneca and Takeda Pharmacutical.