**3.3 Effect of nasally administered HO-1 secreting** *L. lactis* **in murine emphysema model**

HO-1-secreting *L. lactis* were nasally administered to the anesthetized mice 48 hr before instillation with porcine pancreatic elastase (PPE) (**Figure 8**). 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.

#### **Figure 6.**

*Construction of HO-1-expressing vector incorporated into L. lactis. (a) A lactococcal plasmid pNZ8148#2:SEC. (b) A heme oxygenase-1 (HO-1) expression vector (pNZ8148#2:SEC\_mHO1). (c) Vector map of the pNZ8148#2:SEC\_mHO1. Notes: P = nisin A promotor; SPUSP45 = sequence of the signal peptide from the USP45 protein; His-tag = hexahistidine tag; FXa = Factor Xa recognition site; MCS = multiple cloning site; T = terminator; rep = replication gene; and cat = chloramphenicol acetyltrasferase gene.*

#### **Figure 7.**

*Systemic and local HO-1 production after nasal administration of HO-1 L. lactis. Mice (8–9 weeks of age) administered nasally with HO-1 L. lactis were subjected to assess the local (lung) and systemic (serum) HO-1 levels 48 or 72 hr after administration. (a) The lung homogenates from naïve mice receiving either control or HO-1 L. lactis were assessed by immunoblotting. The representative result showed that the nisin-induced HO-1 was confirmed. Adapted from reference [13]. (b) Serum HO-1 levels were assessed using ELISA (MK125, TAKARA Bio Inc., Japan) 48 hr after administration. Results from 5 to 6 mice/group showed a significant increase in HO-1 in both naïve and emphysema models receiving HO-1 L. lactis compared with those receiving control L. lactis.*

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

#### **Figure 8.**

*Protocol of the prophylactic use of HO-1 L. lactis in emphysema model. HO-1 L. lactis was administered 48 hr before instillation of 1 unit of porcine pancreatic elastase (PPE; Elastin Products Co., Inc., USA) in 50 μL of saline. The mice treated with PPE showed progressive destruction of the alveolar structure, leading to emphysematous morphologic deterioration up to day 21.*

On day 21, after PPE instillation, the mice developing pulmonary emphysema were evaluated by pulmonary function test using the flexiVent system (emka TECHNOLOGIES Japan).

*3.3.1 Systemic effect of nasally administered HO-1 secreting* L. lactis

Mice pretreated with 1.0 × 10<sup>9</sup> of HO-1 *L. lactis* showed a significant increase in body weight compared with those pretreated with control *L. lactis* or only saline

#### **Figure 9.**

*Effect of nasal administration of L. lactis on PPE-induced weight loss. Time-course analysis of percent change in body weight after PPE instillation (Day 0) in mice pretreated nasally with 1 × 109 of either HO-1 L. lactis or control L. lactis (Day -2). A significant body weight loss observed in mice pretreated with saline (vehicle only) was not reproduced in mice pretreated with HO-1 L. lactis. The calculated area under the curve of body weight from 5 to 6 mice per group indicated a statistically significant improvement in body weight loss in the HO-1 L. lactis group compared with the control L. lactis or saline group. \* p < 0.05. Adapted from reference [13].*

#### **Figure 10.**

*Local effect of nasally administered HO-1 L. lactis on PPE-induced emphysema mice assessed by in vivo lung function measurements. Mice were treated as described in Figure 8. In vivo lung function tests were performed under anesthesia using a flexiVent system on day 21. The results of lung function measurements of (a) Elastance and (b) Tissue elasticity were shown. Bars indicate the mean ± SD. \* p < 0.05. Adapted from reference [13].*

(*p* < 0.05) (**Figure 9**). Thus, nasal administration of HO-1 *L. lactis* reduced the physiological deterioration caused by PPE.

*3.3.2 Local effect of nasally administered HO-1 secreting L. lactis*

In human clinical trials, the efficacy of candidate drugs for COPD should be primarily assessed by inhibiting lung function deterioration [20]. Therefore, in vivo lung function measurements of mice receiving with or without HO-1 *L. lactis* before emphysema development were assessed using a highly sensitive and reproducible flexiVent system for small animal [21]. The characteristic of an emphysematous lung is reduced elasticity reflecting the hyperinflation and decreased elastic recoil [21]. Consistent with this lung morphologic deterioration, "elastance" (determined by single-frequency forced oscillation technique) and "tissue elasticity" (defined by a small amplitude broadband oscillation technique) were significantly decreased in PPE-induced emphysema mice pretreated with either saline or control *L. lactis*. Fortunately, however, the mice pretreated with HO-1 *L. lactis* showed satisfactory suppression of PPE-induced lung function deterioration (**Figure 10**).

## **4. Conclusions**

This chapter summarizes the potential therapeutics of gmLAB and its application for lung diseases, including COPD. LAB has been widely used as probiotics for health, and to maximize its beneficial effects, gmLAB has been developed. Among several gmLABs, the use of *L. lactis* has been favored because of 1) its generally recognized

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

as safe status, 2) its absence of endotoxins, 3) its easy manipulating property, and 4) its low cost and easy administration. When applied for lung diseases, direct delivery of the therapeutics (gmLAB) to the lungs by intratracheal administration would be favored in terms of efficacy and safety concerns. In addition, the successful attenuation of disease progression in the murine emphysema model by local administration of anti-inflammatory gmLAB would support a further human clinical trial.
