Associations of MMP9 polymorphism with the risk of severe pneumonia in a Southern Chinese children population

Community-acquired pneumonia (CAP) is an infectious lung disease and particularly affects children under 5 years of age [1‐3]. Severe CAP is also a leading cause of childhood morbidity and mortality worldwide, with an estimated 120 million cases per year, of which about 1.3 million cases cause mortality [4]. Children with severe pneumonia often develop various sequelae, including chronic obstructive pulmonary diseases (COPD), bronchiolitis obliterans (BO), pulmonary fibrosis, bronchiectasis, and asthma [5‐7]. The cause of childhood pneumonia is complicated. Viral and bacterial infections were the primary environmental cause of severe pneumonia [8].

The matrix metallopeptidase 9 (MMP9) is a member of the matrix metalloproteinase (MMP) enzyme family, which is defined by the zinc ion in the catalytic domain [9]. The MMP enzyme family plays an essential role in the degradation and regulation of extracellular matrix (ECM) proteins [10]. MMP9 belongs to the gelatinase subtype of MMP [11], and is involved in multiple biological processes including proteolytic ECM degradation [12], cell migration and invasion [13], cell adhesion [14], cell interaction [15], apoptosis [16], and angiogenesis [17]. Notably, circulating MMP9 level has been reported as a biomarker for severe CAP [18], COVID-19 [19], ventilator-associated pneumonia [20] and acute respiratory distress syndrome (ARDS) [21]. Mechanistically, MMP9 may contribute to inflammation by promoting the infiltration of leukocyte and proinflammatory macrophages, as demonstrated in experimental glomerulonephritis [22]. A recent study showed that the skin lesion infiltrating neutrophils in psoriasis overexpressed the MMP9 gene and secreted MMP9 protein. MMP9 activates vascular endothelial cells through MAPK signaling pathways and enhances CD4⁺T cell transmigration in psoriatic pathogenesis [23].

It has been shown that polymorphism in MMP9 were associated with breast cancer risk [24] and chronic venous disease [25]. In addition, MMP9 polymorphism has been suggested to play protective roles in diabetic microvascular complications and pressure ulcers [26, 27]. In summary, MMP9 is associated with inflammatory diseases and is a biomarker for severe pneumonia. However, the associations between MMP9 polymorphism and severe childhood pneumonia need further elucidation.

In order to further investigate the association between MMP9 polymorphism and CAP susceptibility, we conducted a case-control study including a South Chinese population with 1034 cases and 8624 controls to verify the effects of selected SNP. Our results suggest epistatic association of MMP9 rs3918262 SNPs and severe childhood pneumonia.

Community-acquired pneumonia is a common cause of morbidity and mortality in children under 5 years [31]. Although most of the cases are self-limiting disease, around 13% of severe pneumonia cases in PICU unfortunately pass away [32]. Serious sequelae, such as bronchiolitis obliterans (BO), pulmonary fibrosis and bronchiectasis [5‐7], are another burden for those who recover from severe pneumonia. Risk factors of severe pneumonia are numerous, including age, immunodeficiencies, malnutrition, chronic lung diseases, and cystic congenital thoracic malformations [33]. Previous research has documented the existence of gender disparities in community-acquired pneumonia [34]. Consistent with these findings, our cohort also exhibited a gender distinction, as over 50% of the patients were male.

The matrix metalloproteinase (MMP) family plays essential roles in lung organogenesis and the pathogenesis of inflammatory diseases [35]. Dysregulation of MMP contributes to a series of lung tissue damage and disorders such as asthma [36], idiopathic pulmonary fibrosis (IPF) [37], emphysema [38], ARDS [39] and COPD [40]. MMP are also involved in the remodeling of lung after inflammation [41, 42]. Among MMP family members, matrix metalloproteinase MMP9 is enriched in lungs of asthma, IPF and COPD, and also promotes lung remodeling [43]. Associations are also found for MMP9 polymorphisms with lung cancer [44], COPD [45] and asthma [46].

In this study, we first screened the common SNP within MMP9 gene and located 6 candidate SNPs (Table S1). Then we identified rs3918262 and rs17577 as priority SNP by RegulomeDB and FORGEdb analysis (Table 2). Notably, our study showed that the G allele frequency of rs3918262 was significantly associated with increased risk of severe pneumonia (Table 3). No previous study has mentioned the function of this allele. Moreover, rs3918262 showed evidence of a trend towards significance between patients primary or secondary diagnosed as severe or pneumonia (Table 4). Secondary pneumonia was defined as pneumonia caused by non-respiratory reasons [47]. This finding suggests that the minor allele of G of rs3918262 is potentially associated with infections.

From expression quantitative trait loci (eQTL) analysis, we found SNP rs17477 and rs3918262 of MMP9 gene potential regulatory effect on MMP9 or CD40 expression. As CD40 gene is located downstream of the MMP9 gene and in its vicinity, these SNPs may influence the expression of the CD40 gene. In addition, CD40 has been reported to be associated with inflammatory immune diseases such as Kawasaki disease [48], suggesting its potential relevance in regulating pneumonia as well. Most of the alleles exhibit up-regulated effects as the effect sizes were > 0. However, no significant difference was detected in lung tissue. Nevertheless, results from other organs suggest that these SNPs may up-regulate the protein expression of MMP9 or CD40.

Additionally, eQTL, RegulomeDB and FORGEdb results suggest that rs17577, which is located in the coding region of MMP9 gene, had strong regulatory potential on gene expression. Rs17577 was reported to be associated with pediatric asthma [49, 50], breast cancer [51], and ischemic stroke [52] etc. Although the P-value (P = 0.0198) of rs17577 SNP between control and pneumonia group did not meet the threshold (0.00083) of Bonferroni’s correction, this SNP may have important impact on MMP9 expression and potentially was a risk factor of severe pneumonia. It should be noted that no previous study reported about the regulatory functions of the minor allele of G of rs3918262.

Finally, we measure the MMP9 protein levels in the BAL of children with severe or non-severe pneumonia. Results showed that the MMP9 concentrations were up-regulated in severe patients (Fig. 2). In the literature, MMP9 is produced by activated alveolar macrophages and neutrophils, catalyzes the proteolysis of the extracellular matrix and plays a role in leukocyte migration [53, 54]. Taken together with our regulatory results, MMP9 SNPs may contribute to severe pneumonia susceptibility through regulatory properties which may impact MMP9 translational efficiency and protein level. However, these remain to be investigated experimentally in the future. Finally, we demonstrated that protein levels of MMP9 were significantly increased in the BAL of patient with severe pneumonia, suggesting that MMP9 polymorphism may associated with the expression levels of MMP9 in severe pneumonia.

There are some limitations for this study. First, only children were included in our cohort which may reduce the statistical power of the study. Second, the regulatory effects of the SNPs should be validated by subsequent experiments. Finally, only Chinese Han subjects from Southern China were enrolled in this study, and the conclusions should be extrapolated to other ethnic groups with caution.

Nevertheless, this study indicates that the polymorphisms in MMP9 are associated with the susceptibility to severe pneumonia in Southern Chinese children. Future study may elucidate mechanisms of action and reveal whether MMP9 may serve as a potential biomarker or therapeutic target for severe childhood pneumonia.