Epithelial Ablation of Miro1/Rhot1 GTPase Leads to Mitochondrial Dysfunction and Lung Inflammation by Cigarette Smoke

Cigarette smoke (CS) exposure results in lung damage and inflammation through mitochondrial dysfunction. Mitochondria quality control is sustained by Miro1 (Rhot1), a calcium-binding membrane-anchored GTPase by its interaction with PINK1/Parkin during mitophagy. However, the exact mechanism that operates this interaction of mitophagy machinery in Miro1 degradation and CS-induced mitochondrial dysfunction that results in lung inflammation remains unclear. We hypothesized that mitochondrial Miro1 plays an important role in regulating mitophagy machinery and resulting lung inflammation by CS in mouse lung. We showed a role of Miro1 in CS-induced mitochondrial dysfunction and quality control mechanisms. The Rhot1Fl/Fl (WT) and lung epithelial cell-specific Rhot1 KO were exposed to mainstream CS for 3 days (acute) and 4 months (chronic). The cellular infiltration, cytokines, and lung histopathology were studied for the inflammatory response in the lungs. Acute CS exposure showed a notable increase in the total inflammatory cells, macrophages, and neutrophils associated with inflammatory mediators and Miro1 associated mitochondrial quality control proteins Parkin and OPA1. Chronic exposure showed an increase infiltration of total inflammatory cells and neutrophils versus air controls. Histopathological changes, such as pulmonary macrophages and neutrophils were increased in CS exposed mice. The epithelial Miro1 ablation led to augmentation of inflammatory cell infiltration with alteration in the levels of pro-inflammatory cytokines and histopathological changes. Thus, CS induces disruption of mitochondrial quality control mechanisms, and Rhot1/Miro1 mediates the process of CS-induced mitochondrial dysfunction ensuing lung inflammatory responses.


Introduction
Cigarette smoke (CS) leads to mitochondrial dysfunction which is associated with lung inflammation (Sundar, Maremanda, & Rahman, 2019). The dysfunction of the mitochondria accelerates the inflammatory process in lung diseases like chronic obstructive pulmonary disease (COPD) (Ahmad et al., 2015). Normal cellular homeostasis and physiology depends on the regulation of mitochondrial function (Ni, Williams, & Ding, 2015). During cellular damages, the dysfunctional mitochondria are generally eliminated by selective degradation -mitophagy (Lazarou et al., 2015). To sustain the quality control of the mitochondria, Miro1 (Rhot1) serves as an essential element in mitochondrial quality control. Rhot1 (Miro1) is a calcium-binding, membrane-anchored GTPase that is required for the calcium-driven movement of the mitochondria on microtubules, especially during mitophagy (Alshaabi et al., 2021;Sundar et al., 2019;Cloonan & Choi, 2016;. Miro1 also serves a vital role in mitochondrial dynamics by its interaction with Pink1 (PTEN-induced putative kinase 1)/Parkin mitochondrial quality control system by serving as a signal for mitophagy (Bueno et al., 2015;Safiulina et al., 2019). Depolarization of the mitochondrial membrane is responsible for initiating mitophagy in the cells. Following this, the mitochondrial quality control is performed by the stabilization of Pink1 on mitochondrial outer membranes through fission reaction. An E3 ubiquitin ligase, also called as, Parkin is then recruited from the cytosol by the Pink1 for further mitochondrial quality control (Scarffe, Stevens, Dawson, & Dawson, 2014;Sundar et al., 2019). After the recruitment of Parkin, the degradation of a mitochondrial fusion core protein -mitofusin2 (Mfn2) occurs.
This allows clearing off of damaged mitochondria from the cell by protein microtubuleassociated protein light chain 3 (LC3) in the isolation membranes that further initiates the formation of autophagosome by localization of damaged mitochondria and that in turn fuses with the lysosomes to remove the dead mitochondrial cells (Ashrafi & Schwarz, 2013;Sekine & Youle, 2018). The Pink1 and Parkin thereby coordinate together during mitophagy to help regulate mitochondrial degradation. The mitochondrial shape and trafficking are maintained by GTPase Miro1 and Miro2. Thus, together they may serve a crucial role in mitochondrial quality control (Nemani et al., 2018). However, the exact mechanism that operates this interaction of PINK1 and MIRO1 and CS-induced mitochondrial dysfunction that results in lung inflammation in COPD, remains unclear.
We hypothesize that the ablation of Miro1 (Rhot1) from lung epithelial cells would result in the dysfunction of mitochondrial quality control (dysfunctional mitophagy). This may in turn exaggerate the adverse effects of cigarette smoke in the lungs, thereby ensuing CS-induced lung inflammation and mitochondrial dysfunction in lung epithelium Sundar et al., 2019). To demonstrate CS-induced disruption in the mitochondrial quality control mechanisms and the role of Rhot1/Miro1 in mediating the process of CS-induced mitochondrial dysfunction, we utilized the epithelial cell-specific partial (heterozygous) and complete (homozygous) Miro1 knockout (Miro1CC10 KO) mouse models which are to exposed to mainstream cigarette smoke. C57/BL6J mice for 3 days (the acute phase), and 4 months (chronic phase). The role of mitochondrial Miro1 and its impact on the progression of lung inflammation along with mitochondrial dysfunction were studied.

Materials and Methods
Ethical Approval: Institutional Biosafety

Ethics Statement
The study is approved via the laboratory protocols by the Institutional Biosafety Committee (IBC) of the University of Rochester Medical Center, Rochester, NY, All animal experiments Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 10 August 2021 doi:10.20944/preprints202108.0220.v1 5 were exposed to the same time duration of normal air as maintained for the CS exposure Yao et al., 2008).

Collection of bronchoalveolar lavage (BAL)
Mice were injected with 100 mg/kg/bw of pentobarbiturate intraperitoneally and then euthanized. Then cannula was inserted into the trachea of mice and the lungs were lavaged with normal saline (0.6 ml volume; 3 times). The lavage fluid was collected and centrifuged and the separated supernatant samples were stored at -80°C (M. Chen et al., 2017;Rajendrasozhan, Chung, Sundar, Yao, & Rahman, 2010;Wood et al., 2014;Yao et al., 2008).

Total cell count in BAL fluid
The cell pallet of BAL fluid was resuspended in normal saline (0.9% NaCl; 1ml/vol.) and stained by trypan blue (cell staining dye) and total cell count/ml was determined by cellometer.

Differential cell count in BAL fluid
For 3 days (acute) exposure, the differential cell count of immune-inflammatory cells (neutrophils, macrophages, CD4, CD8) was done by BD Accuri flow cytometer in BAL fluid.
For four months (chronic) exposure, differential cell counts to check the cell influx in neutrophils and macrophages were performed on cytospin slides with Diff-Quick staining (Cui, Liu, Ip, Liang, & Mak, 2020;Lim, Kim, Lee, Bae, & Kim, 2018). The Diff-Quick staining involves sequential dipping of the slides into different solutions -fixative agent (methanol, blue), solution 1 (eosinophilic, orange), and solution 2 (basophilic, blue) followed by rinsing and 6 drying. The collected smears are first allowed to dry followed by dipping the slide for one second and repeating it for five times each into a fixative followed by stains 1 and 2. The excess is allowed to drain after each dip. After drying it, the slide is rinsed in distilled water or Weise's buffer (pH 7.2). The tape strip is then stuck to slide (sticky side down) and the excess tape is removed. It is then blotted or allowed to dry in the air for further examination at low power and under oil immersion.

Inflammatory mediators
Differential levels of inflammatory mediators in BAL fluid were assessed for their alteration in the levels using Bio-Plex Pro Mouse Cytokine 23-Plex Immunoassay was run using Luminex. Experiments were done following the manufacturer's instructions and the results were presented as pg/mg protein in the samples.

Measurement of lung mechanics
The mechanical parameters of the lungs like lung resistance, tissue elastance, and static compliance were measured by Flex-ivent apparatus (Scireq; Montreal, Canada). All the mice were weighed and anesthetized by pentobarbital (90 mg/kg; i.p. injection) followed by pancuronium (0.5 mg/kg; i.p. injection). Mice were then tracheostomized and cannulated and the cannula was attached to the rodent ventilator and the ventilator was connected to a computer (Hwang et al., 2011;Phillips et al., 2015). Measurements of all lung mechanical parameters were repeated three times for all mice.

Lung Morphometry and histopathology
Mouse lungs were isolated and inflated with 1% agarose (low melting) and fixed with 4% neutral-buffered paraformaldehyde . Dehydration of the fixed tissue was done followed by encasing it in paraffin and sections were cut by rotary microtome. The lung 7 midsagittal section of each tissue was stained with H&E (hematoxylin and eosin) and the Lm (linear intercept) of airspace and pathological changes were determined in lung tissues.

Statistical Analysis
Statistical analysis was done by one-way (ANOVA), Tuckey's post-hock multiple group comparison test by GraphPad Prism 9 software. Data were shown as mean ± SEM. Significance compared between corresponding Air and CS groups of same genotypes as well as in different genotypes. P < 0.05 is considered significant.

Results
CS-induced inflammatory cell infiltration and differential expression of cytokines were observed in lung epithelial cell-specific Rhot1 deleted mice after 3 days and 4 months of CS exposure.

Three days (acute) exposure
3.1.1. Inflammatory cellular influx in Rhot1 epithelial cell specific KO (Rhot1 +/-CreCC10 and Rhot1 -/-CreCC10) and WT (Rhot1 Flp) mice The total number of cells and macrophage count increased significantly in WT (Rhot1 Flp), Rhot1 epithelial cell specific KO (Rhot1 +/-CreCC10 and Rhot1 -/-CreCC10) mice exposed to CS for 3 days. However, none of the groups showed significant changes in the levels of neutrophils, CD4 and CD8 in response to CS exposure ( Figure 1).

Effect of acute, CS exposure on levels of pro-inflammatory mediators in Rhot1
epithelial cell specific KO (Rhot1 +/-CreCC10 and Rhot1 -/-CreCC10) and WT (Rhot1 Flp) mice 3.1.3. WT (Rhot1 Flp) mice exposed to 3 days CS showed increasing trends of several proinflammatory mediators but the changes between the air and CS group were not found significant. CS exposed Rhot1 epithelial cell specific KO (Rhot1 +/-CreCC10 and Rhot1 -/-8 CreCC10) mice showed significant reduction in the levels of MCP1, IL5, Il-1a, IL-3, IL-9, IFN-γ and TNF-α. The rest of the mediators were not significantly affected ( Figure 2).
WT (Rhot1 Flox) mice exposed to chronic 4 months CS showed a significant increase and nonsignificant increasing trends of several pro-inflammatory mediators. CS exposed WT (Rhot1 Flox) and Rhot1  3.1.8. WT Rhot1 Flox mice exposed to CS showed non-significant increasing trends in resistance and compliance and a decreasing trend in elastance, In contrast, KO (Rhot1 +/-CreCC10 and Rhot1 -/-CreCC10) mice exposed to CS did not show any observable changes in 9 resistance, elastance and compliance ( Figure 4). Airspace enlargement also did not show much appreciable changes though there was a trend in CS exposed mouse lungs ( Figure 5).
3.1.9. Effect on airspace enlargement and pathology of lung tissues in Rhot1 epithelial cell specific KO (Rhot1 +/-CreCC10 and Rhot1 -/-CreCC10) and WT (Rhot1 Floxed) mice Lung sections from chronic 4 months CS-exposed Rhot1 CC10 +/-Floxed Cre mice did not show significant airspace enlargement in comparison with air and CS-exposed Rhot1 fl/fl (WT) mice. However, other histopathological changes such as interstitial foamy macrophages and neutrophils are found in CS exposed mice and the changes are more intensified in Rhot1 CC10 +/-Flox Cre mice ( Figure 6).

Discussion
Chronic inflammation of the lungs is the hallmark in the pathogenesis of COPD, which is primarily caused by cigarette smoking. The smoke of cigarettes contains large amounts of toxic chemicals that affect the cells of the lungs by deleterious nature of tobacco smoke (Nyunoya et al., 2014;Yao et al., 2012). The dysfunction in mitochondria due to injury and morphological alterations are shown to be significantly driven by the CS exposure to the lung cells that includes airway and alveolar epithelial cells, fibroblasts, and airway smooth muscle cells (Aravamudan et al., 2014;Ballweg, Mutze, Konigshoff, Eickelberg, & Meiners, 2014;Hara et al., 2013).
However, the mechanism behind this cellular damage and inflammatory response, and the role of Miro1 in mitochondrial dysfunction upon exposure to CS in lung epithelial cells has remained unclear. In this study, the Rhot1Fl/Fl (WT) and lung epithelial cell-specific Rhot1 KO mice were exposed to CS on acute (3 days) and chronic duration (4 months) to examine the role of Rhot1/Miro1 in the CS-disrupted mitochondrial quality control mechanisms and inflammatory response. The role of mitochondrial dysregulation in lung inflammatory diseases like COPD is known (Mizumura et al., 2014;Wiegman et al., 2015). Regulation of cellular events like fission (fragmentation) and fusion (elongation) process allows mitochondria to rapidly change their shape. MFNs (membrane-anchored proteins: Mfn1 and 2) and OPA-1 (optic atrophy 1) are the major proteins that allow mitochondrial fusion by interacting with other proteins like Pink1 in the mitochondrial membranes (Y. Chen & Dorn, 2013). We analyzed the differential abundance of mitochondrial fission and fusion proteins in lung epithelial cell-specific Rhot1 KO mice through immunoblot analysis. It was found that the levels of OPA1 (a fusion protein) and parkin Further, other cytokines did not show significant trends for any changes. Further, other cytokines did not show significant trends for any changes. It is known that any accumulation of infiltrating cells like neutrophils and macrophages increases along with inflammatory cells are the prime events in the pathological contribution of smoking to lung (Domínguez-Fandos et al., 2012;Gorska et al., 2008). The disruptions in the mitochondria refer to various events that occur at the cellular level in the mitochondria, such as morphological changes, alteration in its metabolic activity, decreased membrane potential and altered mitochondrial superoxide levels and intracellular Ca2+ flux (Cloonan & Choi, 2016;. This may alter the dynamic nature of the organelle. CS exposure causes an alteration in this dysregulation of mitochondria by modifying its function, mitophagy and resulting in ROS production (Ahmad et al., 2015;Hara et al., 2013). ) . This may yield insights into the strategies that focus to pharmacologically restore the role of mitochondrial quality control proteins to treat lung inflammatory conditions like COPD.

Conclusion
Epithelial Miro1 ablation was shown to augment the inflammatory cell infiltration with alteration in the levels of pro-inflammatory cytokines and histopathological changes in mouse lungs ( Figure 7). The study thereby establishes the hypothesis that CS induces disruption in mitochondrial quality control mechanisms, and Rhot1/Miro1 is the key regulator of mitochondrial motility during mitophagy that mediates the process of CS-induced mitochondrial dysfunction.
Chakrapani for technical assistance. We also thank our technical staff Daria M Krenitsky for helping us with tissue sectioning and H&E staining.

Declarations and Disclosures: Conflict of or competing interest statement
The authors have declared that no competing interests exist.  Rhot1 Fl/Fl (WT) and Rhot1 CreCC10 (Rhot1 flp CreCC10 +/and Rhot1 flp CreCC10 +/+ ) mice were exposed to room air and CS (mainstream) for 3 days (acute exposure).
Differential cell count in BAL fluid rom air and CS-exposed mice for 3 days was determined.
Alteration in A. Total cell count B. Macrophages C. Neutrophils D. CD4 and E. CD8, occurred due to CS exposure. Significance compared between corresponding Air and CS groups and all the groups compared with each other irrespective of their exposures. Data are shown as mean ± SEM (n=5 to 13 per group). **P < 0.01, ***P < 0.0001   CreCC10 +/+ ) mice were exposed to room air and CS (mainstream) for 3 days (acute exposure).
Expression levels of pro-inflammatory and inflammatory mediators in BAL fluid rom air and CS-exposed mice for 3 days was determined using Bio-Plex Pro 23-plex cytokine assay.
Significance compared between corresponding Air and CS groups and all the groups compared with each other irrespective of their exposures. Data are shown as mean ± SEM (n=5 to 13 per group). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 The number of total cells in BAL fluid from air and CS-exposed mice for 4 months was determined. B: Lavaged macrophage and C: neutrophil numbers were counted in Diff-Quik stained cytospin slides, which were prepared using BAL fluid. Quantification of macrophages neutrophils expressed as % of total cells in BAL fluid from air and CS exposed mice. CS exposed Rhot1 CreCC10 (Rhot1 CreCC10 +/-) mice showed significant increase in total no of cells and number of neutrophils. Data are shown as mean ± SEM (n=3 to 5 per group).**P < 0.01, ***P < 0.001significant compared with corresponding air exposed mice   CreCC10 +/+ ) mice were exposed to room air and CS (mainstream) for 4 months (sub-chronic exposure). Expression levels of pro-inflammatory and inflammatory mediators in BAL fluid rom air and CS-exposed mice for 4 months as determined using Bio-Plex Pro 23-plex cytokine assay.
Significance compared between corresponding Air and CS groups and all the groups compared with each other irrespective of their exposures. Data are shown as mean ± SEM (n=5 to 13 per group). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Resistance
Elastance Compliance A B C