Study of the Sex Differences in Inflammatory Diseases in Children

Sexual differences in innate immune response have been demonstrated and were mainly attributed to the influence of the sex steroids (1-18). However, recent clinical data revealed significant differences in inflammatory markers between boys and girls suffering from acute and chronic inflammatory diseases (19-23). Sex hormone levels in prepubertal children are particularly low and insufficient to explain the gender differences observed in inflammatory conditions from neonates to the elderly, suggesting the contribution of another mechanism, such as the influence of genes situated on the sex chromosomes and involved in the inflammatory response.

The aim of this work is to evaluate the role of the X chromosome in the sex differences in inflammatory diseases in children. In order to discriminate more precisely the role of the X chromosome relatively to the sex steroids in the sex-specific inflammatory response, some innate immune functions related to X-linked genes will be evaluated in whole blood from prepubertal children of both sexes, suffering from acute inflammatory processes such as pyelonephritis caused by Escherichia coli, pneumonia with pleural effusion caused by Streptococcus pneumoniae or sepsis

Many studies demonstrated immune differences between men and women suffering from acute and chronic inflammatory processes. In cases of acute inflammatory diseases, such as sepsis, females have better prognosis comparing to males (1,24-28).

On the contrary, worse prognosis for women is observed in chronic inflammatory diseases such as asthma or cystic fibrosis (8-10,12,13,29).

Sex-depended inflammatory response was attributed to the influence of sex hormones on the immune system. (2,15-18). However recent studies revealed differences in the clinical outcome but also in inflammatory markers between boys and girls suffering from acute and chronic inflammatory diseases (19-23). Sex hormone levels in prepubertal children are particularly low and insufficient to explain the gender differences observed in inflammatory conditions from neonates to the elderly, suggesting the contribution of another mechanism, such as the influence of genes situated on the sex chromosomes and involved in the inflammatory response.

The aim of this work is to identify the potential X-linked mechanisms responsible for some of the differences between boys and girls in the inflammatory response, making the girls more at risk of developing complications in chronic inflammatory diseases and the boys more at risk of lethal complications in severe acute inflammatory diseases like sepsis. Several genes coding for innate immunity components are linked to the X chromosome such as diapedesis molecule CD99 or TLR pathway proteins genes. (30-33). X chromosome is also highly enriched in genes encoding micro RNAs (miRNAs) involved in the post-transcriptional regulation of gene expression which play a critical role in immune inflammatory response (34-36).

Thus, in order to discriminate more precisely the role of the X chromosome relatively to the sex steroids in the sex-specific inflammatory response, some innate immune functions related to X-linked genes will be evaluated in whole blood from prepubertal children of both sexes, suffering from acute inflammatory processes such as pyelonephritis caused by Escherichia coli, pneumonia with pleural effusion caused by Streptococcus pneumoniae or sepsis. We will also study the correlations between inflammatory and clinical markers of the disease activity to identify prognosis indicators depending on the sex. Additionally, to delineate microbiome contribution, we will study the gut microbiota in stool samples obtained from the recruited patients.

Eligibility Criteria:
Inclusion Criteria of Experimental group :

* Male (XY) and female (XX) aged from 6 months to 7 years old.
* Subject hospitalized either for:

  (1) Urinary tract infection caused by Escherichia Coli, with:
* Temperature ≥ to 38,5°C
* Urinalysis

  * Leukocyte esterase +
  * AND/OR Nitrites +
  * AND/OR pyuria (≥ 100WBC/mm³)
  * AND/OR bacteriuria.
* Urinalysis

  * Clean catch voided urine: \> 10\^4 Escherichia Coli colony form unit (CFU)/mm (urine collection method for children \>3 years old or toilet trained children or by stimulation for children \<3 years old)
  * Transurethral bladder catheterisation: \> 10\^4 Escherichia Coli colony form unit (CFU)/mm³ (urine collection method for children \<3 years old).
  * Suprapubic aspiration: \> 1 Escherichia Coli colony form unit (CFU)/mm³ (urine collection method for children \<3 years old).

    (2) Pneumonia with pleural effusion with :
* Temperature ≥ 38,5°C
* Chest radiography/ultrasound: Pleural effusion
* Streptococcus pneumoniae identified on blood or pleural fluid culture or by PCR

  (3) Sepsis with:
* Documented or suspected infection
* Temperature \< 36° or \> 38.3°C
* Heart rhythm:

  * 2 SD above normal for age
  * 6-23 months: \>180/min
  * 24-71 months: \>140/min
  * 72-84 months: \>130/min
* Respiratory Rate:

  * 6-23 months: \>35/min
  * 24 - 71 months: \>30/min
  * 72-84 months: \>20/min
* WBC:

  * 6-23 months: \>17500/µL or \<5000/µL
  * 24-71 months: \>15500/µL or \<6000/µL
  * 72-84 months: \>13500/µL or \<4500/µL
  * and/or CRP (blood) \> 2SD above normal
* And at least two of the following:

  * PaO2/FiO2 \<300
  * Proven need for \>50% FiO2 to maintain saturation ≥ 92%
  * Need for mechanical ventilation
  * Glasgow score \< 11
  * Urine output \< 0,5mL/kg/h for at least 2h
  * Creatinine:

    * 6-11 months: \>0,4mg/dL
    * 12-23 months: \>0,5mg/dL
    * 24-59 months: \>0,8mg/dL
    * 60-84 months: \>1mg/dL
    * Or creatinine increase more than 0,5 mg/dL
  * Platelet count \<100000/mL
  * Bilirubin \>2 mg/dL
  * Mean arterial pressure (MAP)

    * 6-11 months: \<55 mmHg
    * 12 -23 months: \<60 mmHg
    * 24-59 months: \<62 mmHg
    * 60-84 months: \<65 mmHg
  * SBP less than two SD below normal for age
  * Prolonged capillary refill: \> 5 sec

Inclusion Criteria of Control group :

* Male (XY) and female (XX) aged from 6 months to 7 years old.
* Scheduled surgical intervention for a non-infectious pathology.

Exclusion Criteria:

* Use of antithrombotic drugs (acetylsalicylic acid, thienopyridines, dipyridamol, glycoprotein IIb / IIIa antagonists, vitamin K antagonists, heparins).
* Congenital or acquired immunodeficiency: immunosuppressive drugs, hematopoietic stem cells transplantation, immunoglobulin therapy, extracorporeal membrane oxygenation (ECMO).
* Hemodialysis.
* 48h following cardiac operation of any type.
* Malignant cancer.
* HIV.