**2. Usage of lactic acid bacteria for intratracheal administration**

#### **2.1 Construction of genetically modified lactic acid bacteria (LAB)**

There have been various LABs constructed for specific target therapy and/or monitoring the LAB dynamics after administration in the animal/human body. Lactococcus (L.) lactis NZ9000 for nisin regulated target gene expression system (MoBiTec, Goettingen, Germany) was used for these purposes. The genetically modified *L. lactis* was grown under the anaerobic condition at 30°C in M17 broth (BD DificoTM) overnight. The target gene expression was induced by adding 1.25 ng/mL of nisin (MoBiTec). Of these gmLABs, a green fluorescent protein (GFP)-fusion target gene expressing LAB enables researchers to monitor the levels of target gene expressions [19]. **Figure 1** shows the vector constructed for monitoring the time-dependent migration after nasally administering *Lactococcus lactis* that express/produce GFP over time.

The GFP-expressing *L. lactis* was cultured, and further time course was monitored for expression levels of GFP. Three hours after adding nisin (1.25 ng/mL), the cultured/induced GFP-expressing *L. lactis* was visualized under fluorescent microscope observation (**Figure 2**).

#### **2.2 Airway migration of nasally administered** *L. lactis*

GFP-expressing *L. lactis* were nasally administered to the anesthetized mice. A total of 50 μL of saline containing 1.0 × 109 of *L. lactis* was dropped into the nares and migrated to the lungs through stable nasal breathing.

*Intratracheally Therapeutic Option for COPD: A Potential Usage of the Therapeutic Microbe… DOI: http://dx.doi.org/10.5772/intechopen.106491*

#### **Figure 1.**

*Construction of GFP-expressing vector incorporated into the LAB. (a) lactococcal plasmid pNZ8148#2:CYT. (b) A green fluorescent protein (GFP) expression vector (pNZ8148#2:CYT\_GFP). (c) Vector map of the pNZ8148#2:CYT\_ GFP. Notes: P = nisin A promotor; His-tag = hexahistidine tag; FXa = Factor Xa recognition site; MCS = multiple cloning site; T = terminator; rep = replication gene; and cat = chloramphenicol acetyltrasferase gene.*

#### **Figure 2.**

*Image of GFP-expressing Lactococcus lactis after induction with nisin. Genetically modified Lactococcus (L.) lactis were cultured and further induced with nisin for expression of a specific protein. The high-power field image of L. lactis showing diplococci morphology with a green signal derived from the GFP expression vectorincorporated system was visualized using a fluorescent microscope (BZ-X800; Keyence, Japan).*

As shown in **Figure 3**, visualized GFP signal was time-dependently moved from the central lesion to the peripheral lesion of the lungs. Finally, the GFP signal was cleared from the lungs 96 hr after administration. Notably, at the same time of 96 hr, there was still an apparent GFP signal in the trachea, indicating 1) the high affinity of *L. lactis* for tracheal epithelium and 2) the potential usage of *L. lactis* as a carrier of airway mucosal vaccination.

#### **2.3 Systemic effect of nasally administered** *L. lactis*

**97** Potential systemic influences after administering *L. lactis* would be body temperature, body weight, and eating behavior. Of these, time-course analysis of percent

#### **Figure 3.**

*Time-course analysis of ex vivo fluorescence images of removed lungs after administering GFP-expressing* L. lactis*. (a) Mice (8–9 weeks of age) administered nasally with 1.0 × 109 of GFP-expressing* L. lactis *under anesthetized with pentobarbital sodium (30 mg/kg) were euthanized at an indicated timepoint of (b) 24 hr, (c) 48 hr, and (d) 96 hr. The removed lungs were observed under IVIS (In Vivo Imaging System, Perkin-Elmer) with (right panel) or without (left panel) fluorescence excitation. GFP signal visualized in right panel at each time point appeared in the central lesion (trachea and hilar area of the lungs) at 24 hr (b), moved to the peripheral lesion at 48 hr (c), and cleared from the lungs at 96 hr (d).*

#### **Figure 4.**

*Change in body weight after nasal administration of L. lactis. Time-course analysis of percent change in body weight in mice (8–9 weeks of age) administered nasally with 0, 5 × 108 , 1 × 109 , or 5 × 109 of L. lactis. Results showed that a significant body weight loss was observed in mice treated with 5 × 109 of L. lactis. The calculated area under the curve of body weight from 3 to 4 mice per group indicated a statistically significant body weight loss in 5 × 109 of the L. lactis group compared with the saline group. \* p < 0.05. Adapted from reference [13].*

#### **Figure 5.**

*Analysis of lung microbiota 14 days after nasal administration of L. lactis. Mice (8–9 weeks of age) administered nasally with 0, 5 × 107 , 5 × 108 , or 5 × 109 of L. lactis were euthanized and collected bronchoalveolar lavage (BAL) fluids 14 days after administration. The analysis of the 16S rRNA gene (V3-V4 region) was amplified and subjected to next-generation sequencing (3 mice per group).*

*Intratracheally Therapeutic Option for COPD: A Potential Usage of the Therapeutic Microbe… DOI: http://dx.doi.org/10.5772/intechopen.106491*

change in body weight showed the safety concern of mice (8–9 weeks of age) administered nasally with over 1 × 109 of *L. lactis*. As shown in **Figure 4**, the calculated area under the curve of body weight from 3 to 4 mice per group indicated a statistically significant body weight loss in 5 × 109 of the *L. lactis* group compared with the saline group. Based on these results, the optimized amount of nasal administration of *L. lactis* was set to less than 1 × 109 per body at one time.

#### **2.4 Local effects of nasally administered** *L. lactis*

Another concern after nasally administering *L. lactis* would be a potential alteration of lung microbiota. As shown in **Figure 5**, intratracheal administration of up to 5 × 10<sup>9</sup> of *L. lactis* would show no statistical significance in 1) Bacteroidetes to Firmicutes ratio and 2) the composition of the microbiota belonging to Bacteroidetes or Firmicutes compared with those observed in control (saline) group.
