Mast cells participate in regulation of lung‐gut axis during Staphylococcus aureus pneumonia

Abstract Objectives The lung‐gut axis is known to be involved in the pathogenesis of Staphylococcus aureus pneumonia. However, the underlying mechanisms remain unclear. We examined the role of pulmonary mast cells (MCs) in the regulation of the lung‐gut axis during S. aureus pneumonia. Materials and Methods We created a mouse model of S. aureus pneumonia using MC‐deficient mice (Kit W‐sh/W‐sh) and examined the level of inflammation, bacterial burden, expression of cathelicidin‐related antimicrobial peptide (CRAMP) and composition of the gut microbiota. We further evaluated anti‐bacterial immunity by administering bone marrow MCs (BMMCs) or CRAMP into the lungs of Kit W‐sh/W‐sh mice. Results After S. aureus challenge, the MC‐deficient mice, compared with wild‐type (WT) mice, displayed attenuated lung inflammation, decreased expression of CRAMP, higher bacterial lung load and disturbance of the intestinal microbiota. Adoptive transfer of BMMCs into the lung effectively reconstituted the host defence against S. aureus in Kit W‐sh/W‐sh mice, thus resulting in recovery of S. aureus pneumonia‐induced intestinal dysfunction. Similarly, exogenous administration of CRAMP significantly enhanced anti‐bacterial immunity in the lungs of MC‐deficient mice. Conclusions This study provides evidence for the involvement of MCs in the regulation of the lung‐gut axis during S. aureus pneumonia.


| INTRODUC TI ON
Staphylococcus aureus is one of the common pathogens in hospital-and community-acquired pneumonia. 1 In addition to the symptoms including headache, fever, dyspnoea and cough, patients with pneumonia often present with gastroenteritis-like discomforts such as vomiting and diarrhoea, especially in young children. 2 Increasing evidence indicates that the intestinal microbiome and mucosal tissues broadly affect the progression of multiple diseases. 3 The lung-gut axis, which represents the interaction between lung immunity and intestinal microbiota, 4 is thus an area of interest, though the underlying mechanisms remain poorly understood.
Mast cells (MCs) are particularly abundant at host-environment interfaces, such as the skin and the respiratory and gastrointestinal tracts. Because of their location, MCs have been hypothesized to act as sentinel cells that sense pathogen attacks and initiate a protective immune response. 5 However, knowledge of the role of MCs in the host defence against S. aureus is limited. Recent studies have demonstrated that MCs can be activated by S. aureus and exert antimicrobial activity. 6,7 But, anti-S. aureus mechanisms of MCs have not yet been demonstrated in the lung. The lung is the major target tissue of diverse inhaled microbial pathogens, including S. aureus. To combat them, the respiratory system is armed with diverse mechanisms of innate mucosal immunity, including the secretion of antimicrobial peptides. 8,9 Cathelicidins show anti-bacterial activity against both Gram-positive and Gram-negative bacteria through destruction of bacterial membrane. [10][11][12] Cathelicidin expression is upregulated in the airways during bacterial infection 13 and has been detected in alveolar macrophages, neutrophils and airway epithelial cells. 10,14,15 There has been increasing interest in the role of cathelicidin-related antimicrobial peptide (CRAMP) during bacterial pneumonia.
C-kit receptor plays crucial roles in the differentiation, proliferation and inflammatory reaction of MCs. 16,17 Our prior work has shown that MCs released inflammatory factors (eg, tumour necrosis factor (TNF)-α) and CRAMP in response to S. aureus, through stimulation of c-kit receptor and its downstream molecules. 18 In this study, using MCdeficient mice (Kit W-sh/W-sh ) with defective c-kit receptor, we evaluated the diversity of the intestinal microbial community in mice with S. aureus pneumonia and determined the role of MCs in regulation of the lung-gut axis. We showed that MC deficiency impaired lung inflammation and aggravated the imbalance in the gut microbiota upon S. aureus infection. Adoptive transfer of bone marrow mast cells (BMMCs) into the lung largely reconstituted the host defence against S. aureus in both the lung and the gut, thus demonstrating a critical role of MCs in the immune response through regulation of the lung-gut axis.

| Animals
Kit W-sh/W-sh mice on a C57BL/6 background were kindly provided by Harvard Medical School. 19 Age-and sex-matched wild-type (WT) mice were used as controls. All animal experiments were approved by the Animal Care and Use Committee of The Second Affiliated Hospital, Zhejiang University School of Medicine.

| Bacterial culture
A clinical isolate of S. aureus was grown aerobically overnight at 37°C in a shaking incubator. 18,20 S. aureus in the mid-logarithmic phase was resuspended in phosphate-buffered saline (PBS). The numbers of bacteria was quantified according to the OD600-based bacterial growth curve and verified with colony-forming unit (CFU) assays. 20

| BMMCs culture
Bone marrow mast cells were generated from the femurs of C57BL/6 mice and maintained in the presence of 10% pokeweed mitogen-stimulated spleen-conditioned medium, as described previously. 18

| A mouse model of S. aureus lung infection
Eight-week-old C57BL/6 and Kit W-sh/W-sh mice were used to establish an S. aureus pneumonia model. 22 In brief, 40 μL of S. aureus (5 × 10 7 CFUs) was inoculated intratracheally into anesthetized mice. Non-infected control mice were administered with an equal volume of PBS intratracheally. Lung specimens were weighed and homogenized in 1 mL PBS to determine bacterial load and production of cytokines and CRAMP.

| Reconstitution of MCs in Kit W-sh/W-sh mice
Kit W-sh/W-sh mice (5-6 weeks old) were injected with 1 × 10 7 BMMCs in 200 μL PBS via the tail vein. The mice treated with PBS were used as controls. After 3 weeks, the reconstituted mice were used to build S. aureus pneumonia model, and tissues were harvested for analysis after 24 hour post-infection. 23

| CRAMP treatment
The effects of CRAMP on S. aureus pneumonia were examined through intratracheal administration of 20 mg/kg CRAMP (GL Biotech, Shanghai, China) in 40 μL PBS to mice for four consecutive days. 24

| Histological analysis
Mouse lung specimens were fixed in 10% paraformaldehyde, embedded in paraffin, cut into 4 μm-thick sections and stained with haematoxylineosin (H&E). The stained sections were reviewed for morphology under a photomicroscope (Leica, Heidelberg, Germany). The neutrophil infiltration and inflammatory scores of the lung samples were determined as described previously. 25,26 In brief, for evaluation of lung neutrophils, three lung sections from each mouse were analysed and three randomly selected high-power fields (HPF) were examined for each lung section, the average value of cells/HPF for each mouse was determined by summation of all numbers divided by 9. The inflammatory levels were semi-quantitatively determined as below: peribronchial/peribronchiolar inflammation 0-4, perivascular inflammation 0-4 and alveolar inflammation 0-2. Each lung section was given a total inflammatory score (maximum score of 10).

| Immunofluorescence staining
Paraffin-wax sections of mouse lung tissues were placed on glass slides, and non-specific antibody binding was blocked by incuba- were incubated with the above antibodies overnight at 4°C, then incubated at 37°C for 30 minutes. DAPI was used to stain the nuclei before viewing by confocal microscopy (Olympus, Tokyo, Japan).

| Statistical analysis
Data are expressed as mean ± standard error of mean (SEM), unless otherwise stated. Differences between two or multiple groups were analysed with Student's t test or one-way ANOVA as appropriate.
CFUs in lung tissue were compared using the Mann-Whitney U test.
A P value <0.05 was considered statistically significant. All calculations were performed in GraphPad software (Version 5.01).

| MC deficiency decreases the inflammatory response to S. aureus infection
We evaluated the effect of MCs on the inflammatory response to  Figure 1D). Confocal microscopy examinations showed that S. aureus stimulation upregulated the expression of lung MC-derived CRAMP in WT mice, but weak expression of CRAMP was found in Kit W-sh/W-sh mice ( Figure 1E). Meanwhile, Kit Wsh/W-sh mice had significantly higher numbers of S. aureus in their lungs after infection, compared to WT mice ( Figure 1F).

| Kit W-sh/W-sh mice display impaired intestinal microbiota after S. aureus lung infection
To explore the influence of MCs on the intestinal microbiota,  Figure 2B), which accounted for approximately 2% of the total faecal microbiota in uninfected WT and Kit W-sh/W-sh mice, but raised to approximately 6% and 15%, respectively, in infected mice.

| Adoptive transfer of BMMCs reconstitutes host defence against S. aureus infection in Kit W-sh/W-sh mice
We performed an adoptive MC transfer experiment to determine whether the susceptibility of Kit W-sh/W-sh mice to S. aureus was mainly attributed to MC deficiency. Treatment of WT-derived BMMCs led to significantly elevated levels of IL-6, TNF-α, KC, MIP-2 and CRAMP in KitW-sh/W-sh mice upon infection ( Figure 3A). Correspondingly, MC-reconstituted Kit W-sh/W-sh mice exhibited markedly improved bacterial clearance of S. aureus ( Figure 3B). Furthermore, sequencing analysis of the microbiota also indicated that the imbalance in the intestinal flora after S. aureus infection was largely reversed in MC-reconstituted Kit W-sh/W-sh mice ( Figure 4A-B).

| Exogenous CRAMP treatment significantly ameliorates S. aureus pneumonia
Exogenous CRAMP was intratracheally administrated into the mice for 4 consecutive days. Although the lung-tissue levels of the proinflammatory cytokines IL-6, TNF-α, KC and MIP-2 were not significantly affected by CRAMP treatment (Figure 5A), the bacterial load was obviously decreased by CRAMP treatment in both WT and Kit W-sh/W-sh mice ( Figure 5B).

| D ISCUSS I ON
Staphylococcus aureus is one of the most common causative pathogens of pneumonia. 28 Although pneumonia is known to be a lung infection, many studies have shown that pneumonia is also associated with substantial extrapulmonary effects, including intestinal inflammation, apoptosis and injury. 29,30 Mucosal tissues, including those in the res-  36,37 In this study, using Kit W-sh/W-sh mice, we revealed that MC-deficient mice were significantly more susceptible to S. aureus lung infection than WT animals, accompanied by a greater imbalance of the intestinal flora. We further carried out adoptive transfer of BMMCs into Inflammation is an integral part of the reactions of the innate immune systems against micro-organisms. 38 We recently showed that the activated MCs contributed to a series of inflammatory actions to clear the pathogen in a mouse model of skin S. aureus infection. 18 The host inflammatory and antimicrobial responses against S. aureus were impaired in Kit W-sh/W-sh mice, which suggested that MCs may act as an immune booster in the lung. Consistently, our previous data showed that MCs released multiple inflammatory cytokines directly upon S. aureus infection by means of the c-kit-activated phospho- In summary, our results demonstrated that lung MCs are involved in regulation of the lung-gut axis during S. aureus pneumonia.
This study thus provides a potential clinical strategy focused on MCs for patients with S. aureus pneumonia accompanied by severe gut symptoms.

ACK N OWLED G EM ENTS
This work was supported by grants from the National Natural Science Foundation of China (81770008 and 81570004).

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.