extfieldsofstudy: Medicine Biology

text:
Tumor progression mechanisms: Insights from the central immune regulation of tissue homeostasis

Knowledge of the mechanisms underlying the spread of cancer at the cellular and molecular levels is expanding rapidly. However, the central regulators governing the initiation and the rate of tumor growth remain poorly established. The fundamental principles of innate and adaptive immunity may explain how immune cells generate a specific response to tumor tissue. In the current review, the functional features of the immune system that contribute to the maintenance of normal tissue homeostasis, as well as their disruption in malignant transformations, were analyzed. Experimental and clinical studies previously demonstrated the involvement of regulatory T-cells in the process of tumor metastasis in a tissue-specific manner. An understanding of the cross talk between lymphoid and tumor cells may provide an insight into cancer evolution in terms of the mechanisms of T-cell competency formation. Elucidating the mechanisms of tumor progression via central immune regulation has implications for the development of novel therapeutic agents that target immune checkpoints.

Introduction
Elucidation of the mechanisms underlying the metastasis of solid tumors may improve patient outcomes, as the majority of cancer-associated mortalities are due to tumor progression and dissemination (1). Previous studies have investigated the role of immunity in tumor biology; however, the immune-mediated maintenance of tissue homeostasis (TH) in cancer has not been thoroughly explored (2)(3)(4). Tumor progression may be influenced by the immune system (2,3). However, the role of tissue-specific factors in the outcome of adaptive immune responses remains unclear (4). Advances in molecular biology have allowed the investigation of the mechanisms involved in intercellular interactions. Consequently, there is an increased understanding of the factors involved in these interactions, allowing a comprehensive description of tissue development to be made. A number of immune cells, cytokines, growth factors and enzymes create a favorable microenvironment for maintaining a steady state in tissues (5,6). However, which of these are essential in maintaining a favorable tissue microenvironment remains unknown. The interchange of signals at the tissue level is maintained by constant synthesis of various short-lived molecules, including cytokines, integrins and selectins, and exosomes (7,8). TH is therefore a complex process. The present review attempted to analyze the functions of the immune system, not including its protective role against infection, and establish whether it has a morphogenetic or regulatory function in maintaining TH (9,10). Consequently, the present review focused on regulatory T cells, due to the tissue-specific manner these cells operate in (11,12). Although T cells reside in tissues, they are functionally monitored by the thymus, and provide feedback on TH from the peripheral to the central immune system by their homing capacity (13). Elucidating the central immune regulation of tumor tissue

Tumor progression mechanisms: Insights from the central immune regulation of tissue homeostasis (Review)
evolution may aid in the development of novel immune therapies.

Central immune regulation in maintaining homeostasis of normal tissue
The immune system detects pathogen invasion, fights against infection, prevents malignant transformations and contributes to permanent tissue renewal and remodeling following damage to the tissue (10,14). The thymus is a central immune organ that serves a principal role in post-natal human life by maintaining the development and homeostasis of T cells (15,16). The regenerative capacity of the thymus undergoes a significant decline in the postnatal period compared with its prenatal activity; however, it continues to perform a pivotal role in T-cell arrangement in adulthood (17). Stem cells of the thymic epithelium are constantly generated throughout the lifespan of a human and their pool is dynamically regulated by signals from the periphery in response to tissue requirements (18). The thymus generates clones of regulatory cells during the process of T-cell development, which occurs due to the recognition of self-antigens (19). Thymically derived regulatory cells (T-regs) are considered to serve important roles in maintaining and re-establishing of normal TH (20,21).
The immune system has a functional presence in every tissue and organ, as immune cells have the unique ability to travel between compartments (11). Each organ has a microenvironment that participates in feedback mechanisms in which dendritic cells present tissue major histocompatibility complex (MHC) antigens for recycling lymphocytes (22). The continuous recycling of lymphocytes from the blood into tissues is a highly sophisticated bi-directional process that reflects the homing capacity of migrating cells (19). The migrating lymphocytes patrol the tissues, thus maintaining homeostasis by a directional transfer of information, which is beneficial for tissue development (7,10,23). The destination of the lymphocytes is predetermined by T-cell receptors (TCRs), which are located on the surface of migrating cells that interact with the appropriate ligands of high endothelium venules for penetration into the tissue (12). TCRs are designed to recognize different MHC molecules, which are presented by the majority of cells in the body (24). Using specific receptors, lymphocytes assess the antigenic immunity in body tissues, and the binding specificity of the TCRs is important in the process of recognition (25,26). Consequently, the movement of lymphocytes into the lymphatic system and blood circulation, as well as their migration into the tissue, is an organized process (11). The endothelial cells of capillaries are not passive membranes that follow physical and chemical laws, rather they select for active traffic into tissues by target-binding to the receptor by antigen-antibody interaction (12). T-lymphocyte migration directed by TCRs is conducted by ligand-integrin interactions with adhesive molecules of blood vessel endothelium (27). Immune surveillance of peripheral tissues by T cells is performed in response to homeostatic chemokines that are constitutively expressed in healthy tissues to recruit lymphoid cells in certain tissue sites (28). Tissue disturbance due to injury or inflammation results in the secretion of cytokines, triggering the hyperactive expression of specific integrins on the surface of endothelial cells. The lymphocytes connected to the integrins adhere to the endothelium, enter between adjacent endothelial cells and exit the bloodstream (27). Simultaneously, lymphocytes, macrophages and other lymphoid cells, including neutrophils and monocytes, penetrate through the intercellular gaps and re-enter the circulation. Lymphatic tissue is constantly renewing its cell population in order to continuously recycle lymphocytes (14,29). Recirculating lymphocytes are a major part of the population of small lymphocytes, the majority of which are T-lymphocytes (30). T-lymphocytes are characterized by their rapid migration, which enables them to constantly move between the blood and peripheral organs (31,32). As such, small lymphocytes are an extremely mobile cell population. Due to their mobility, lymphocytes are able to penetrate into intercellular spaces (33). Subsequent to passing through the vessel endothelium, lymphocytes may interact with intercellular matrix proteins and tissue cells. The complex interactions among lymphocytes, residing immune cells, stromal components and tissue-specific factors influence the outcome of the immune responses at the tissue level (14). As a result, all functioning tissues have active lymphocytes located around postcapillary venules (11,34). The zone of interaction between endothelial cells, lymphocytes and tissue structures is termed the immune regulatory zone, or the immune regulatory compartment of an organ (34,35). Previous studies demonstrated that the majority of tissue lymphocytes are T-lymphocytes, which originate from the double-positive helper/suppressor population, and are currently defined as the regulatory population (13,36). Through a homing mechanism, T-lymphocytes undergo a settling stage in the tissue, where they acquire their immunological specificity (11).
The thymus is an important site for the development of T-regs during the positive selection process of clones that are tailored to the tissue in which they complete differentiation (37). Following this process, T-regs join the lymphatic system and blood circulation. Circulating lymphocytes form a pool of cells carrying receptors that are specific for the particular tissues that created this clone (11). Once the migration process has started, a functional complex that includes an adjustable tissue and regional lymph nodes is formed. It provides a selectivity of migration, termed homing (12). Such selectivity is associated with effector lymphocytes acquiring novel properties dictated by the local tissue microenvironment (38,39). Therefore, any regenerating/renewing healthy tissue that is non-inflamed drives the preferential recruitment of a highly restricted repertoire of specific T-regs for its continued development (13).
One issue that remains is concerned with the manner in which recirculating T-cells perform the morphogenetic functions of the immune system. Cell-to-cell interactions assist multicellular organisms to function as a stable system (40). Informational support by the transfer of genetic material in the form of exosomes to developing tissue is an immunoediting function of T-cells (41), although the transmission mechanism of information pertaining to regeneration has not been fully elucidated. Previous studies have indicated that cells can communicate via the direct exchange of genetic patterns in the form of exosomes and apoptotic bodies (7,40,42). The processes of cell-to-cell communications via exosomes are a potential driving factor of phenotypic changes and cellular plasticity during tissue regeneration (43). Therefore, specific genetic messages are designed to be embedded into accepting cells for their differentiation with subsequent tissue development. The required level of exosomes is provided by cell activation and apoptosis, which underlies the effector mechanisms of immune cell action (44). The genetic information contained within exosomes in the form of DNA or RNA can influence the target cell by triggering activation, differentiation or even by promoting apoptosis (40). In the immune system, exosomes from T cells can directly fuse with accepted tissue cells, releasing relevant functional information (45). The involvement of lymphocytes in the regulation of tissue development has clearly been demonstrated by their presence in the microenvironment of differentiating hematopoietic cells of the bone marrow (46). Furthermore, T-regs have been shown to serve an important role in maintaining the homeostasis of skin and various mucosal surfaces, from which epithelial tumors have been identified to form (13,47).
In summary, any functioning tissue is a highly organized peripheral division of the immune system with a stable contingent of residing cells and a mobile component comprised of recycling regulatory T cells that are functionally monitored by the thymus. These T cells provide a feedback reaction to the thymus as a central part of the immune system. T-regs actively participate in the generation of adaptive immune responses. The processes of the migration and recirculation of immune cells, and their active interactions with other tissue structures ensure the maintenance of TH (Fig. 1).

Immune regulation in case of tumor tissue manifestation and metastatic spread
One topic of note is what happens to the highly sophisticated hierarchy of TH regulation in the extreme case of its imbalance, i.e., during malignant transformation of tissue. Since tumor cells originate from normal ones, the key principles of immune regulation are preserved in the transformed tissue. Therefore, immune mechanisms that regulate tumor tissue function may be the same as in cases of normal non-metastatic tissues (48).
When tumors develop, the new tissue evolves and the metastatic potential of the rapidly dividing cells (cellular clones) constantly increases. Clinically, metastasis is the last stage of tumor progression. Prior studies revealed that the process of cancer spreading is not random, and follows domestic rules and mechanisms in the 'host-tumor' system (49)(50)(51). This was confirmed when certain cancer types elected to metastasize over their preferred target tissues (52). Previous studies have provided supporting evidence of the importance a common mechanism involved in the regulation of tumor progression: i) Tumor cells acquire metastatic properties prior to migration from the site of the primary tumor (53); ii) the systemic circulation of tumor cells may subsequently remain latent for an extended period of time (54) and the presence of tumor cells in the blood is not indicative of metastasis (55,56); iii) the formation of metastatic niches in the form of restructured stroma in tissues distant from the site of the primary tumor begins prior to the dissemination of malignant cells (2,57); and iv) the enhanced vascularization of blood and lymph vessels in tumors contributes to the process of metastasis (58,59). Clonal polymorphism, genetic instability of tumor cells and a hypoxic microenvironment contribute to the expression of multiple angiogenic factors in malignant tissues (60)(61)(62). Nevertheless, it has been postulated that stimulation of vessel growth in tumors occurs via the same mechanism as that in normal tissue (63). As such, blood vessels treat the tumor as an extra cell mass, which requires nutrition and elimination of the products of tissue metabolism (64). The aforementioned process supports the hypothesis that the influence of the host on the formation of the tumor microenvironment is a factor that determines when the metastatic process is initiated as the formation of metastatic niches begins before the spread of malignant cells, and neoangiogenesis contributes to the process of metastasis. However, the central regulatory mechanisms that activate selected tumor cells for metastatic initiation remain poorly defined.
T-regs with the phenotype cluster of differentiation (CD)4 + CD25 + forkhead box P3 + (FOXP3 + ) imitate mediators of immune editing (surveillance) (65)(66)(67). Novel immune checkpoint inhibitors recognize that T-regs serve a key role in tumor development (68). Therefore, elucidating the associations of T-regs and tumor cells may provide insight into the modulation of the host response to the malignant transformation of tissues (66). T-reg density within tumor lesions and the recurrent imbalance of its contents in the blood has been associated with the clinical outcome of patients in a number of types of cancer, including breast, cervical, endometrial, ovarian, colorectal and pancreatic cancer (69)(70)(71). Tumor-specific T-regs may originate in the thymus during T-cell development and are recruited to the tumor lesion in a preferential manner from the diverse systemic pool of T-regs (19,48).
The phenotype, differentiation status and function of regulatory immune cells depend on the anatomical compartment in which the cells reside. Even immune cells that originated from the same precursors, but which reside in different tissues, have diverse functions as they are instructed by different, organ-specific factors (11). Consequently, different types of tissue have different antigens that activate the immune system and generate regional immune responses, which is associated with the relative stability of the tissue. In cases of tumor transformation due to immune escape mechanisms, the immune system may maintain homeostasis in the newly-formed anatomical site, i.e., the tumor. Therefore, the immune system allows the tumor to grow rather than destroy it (65,72), suggesting a mechanism of how tumor tissue development, and progression may coexist with the normal function of an organ (2,64). Tumor cells are constantly exposed to immune cells during each phase of the metastatic process, suggesting that the immune cells may restrict their development (73). However, previous studies have revealed that tumor-infiltrating immune cells may instead promote the metastatic cascade (2,3,72). T-regs may serve a role in increasing the number of surviving tumor cells in circulation and metastatic sites; however, the precise mechanisms underlying this process remain unknown (2).
The thymus orchestrates the arrangement of T-cell clones and predetermines the function of T-cell populations on the periphery (27,74). A prior study that observed changes in the thymus during experimental carcinogenesis revealed that the development of malignant tumors in the colons of rats was correlated with significant fluctuations in the morphology of the thymus, highlighting the role of central immunological regulation in tumor biology (75). These variations were manifested in the differentiation and apoptosis of thymus cells, as well as changes to the components in the microenvironment of the thymus cells. Notably, significant transformations in the thymus, including an increase in the number of thymic cells, active T-lymphocytes and changes in the microenvironment were observed during the precancerous stage in the intestinal mucosa (75). The aforementioned study reported that colonic cancer had a greater impact on the thymus morphology. It manifested in the elevation of proliferation and differentiation of thymocytes into mature forms with their subsequent traffic to the colonic mucosa (75). Experimental data regarding the increased egress of mature thymocytes and their migration into peripheral circulation are consistent with clinical data reporting an increased T-reg lymphocyte population in the peripheral blood of patients with solid tumors including melanoma and breast, lung, gastric, ovarian, head and neck, tumors (65,70,76). Furthermore, T-reg lymphocytes have been detected in lymph nodes containing micrometastases, but not in healthy lymph nodes (77). The detection of T-reg lymphocytes and the formation of new lymphatic vessels precede the detection of metastatic lesions in the regional lymph nodes (78). T-lymphocyte clones migrate and infiltrate a solid tumor (70). According to numerous studies, they are mainly represented by the same regulatory thymic subpopulation of lymphocytes, which has been discussed previously (48,79,80).
The process of malignant transformation of tissue is accompanied by the formation of T-lymphocyte clones designed for that tissue. These lymphocytes begin to differentiate in the thymus and complete the differentiation process in the tumor tissue, which is actively involved in the generation of immune signals required to maintain tissue development (81). Consequently, the tumor tissue may be considered as a peripheral compartment of the immune system whose regulatory zone includes vessels, endothelial cells, circulating and settled pools of T-reg lymphocytes, tumor cells and components of the extracellular matrix. Therefore, the affected organ consists of tumor and normal tissues. Each of these tissue types has a zone infiltrated by T-reg lymphocytes designed for this tissue type. There are clones for tumor and normal tissues in the peripheral circulation of an organism with cancer. Phenotypically these clones are similar, all being T-reg CD4 + FOXP3 + cells (79,82). However, they differ in the direction of migration to the appropriate tissue, which expresses its own unique set of histocompatible antigens (Fig. 2).
An increased number of T-regs in tumor tissues is associated with a higher degree of differentiation and an improved prognosis (69)(70)(71)76), suggesting that T-regs serve a role in the pace of tumor progression. A previous study in patients with breast cancer assessed T-reg lymphocytes profiles in samples of tumor tissue following the administration of neo-adjuvant chemotherapy, and a significant reduction in T-regs among infiltrating tumor tissue lymphocytes was detected (83). This suggests the possibility of using T-reg lymphocytes as a prognostic marker for response to chemotherapy and demonstrates a direct association between tumor regression and a reduction in the population of T-reg lymphocytes (70,84,85).
Novel immunotherapies may have a greater impact on T-reg lymphocyte subpopulations than traditional chemotherapy agents (68,86). The clinical application of targeted antitumor drugs against cytotoxic T-lymphocyte associated protein 4 and programmed cell death 1 lymphocyte receptors, termed immune checkpoint inhibitors, has demonstrated efficacy in a subset of patients with aggressive solid tumors, including disseminated melanoma, renal cell carcinoma and non-small cell lung cancer (86,87). Furthermore, patients demonstrated partial or complete tumor regression following the administration of the aforementioned immune therapies, which persisted following the cessation of therapy, which was renewed as an identical treatment if disease progression occurred (87). Novel immunotherapies have increased the potential available targets of anticancer drugs, providing insights into the mechanisms underlying tumor development and progression. Preceding studies have identified novel agents that target lymphocytes and alter the immunological microenvironment Lymphocytes begin to differentiate in the thymus and complete their differentiation at the periphery tissue compartment where they migrate due to their homing capacity. As a result, the organ affected by the tumor consists of tumor and normal tissues infiltrated by T-reg lymphocytes designed for the particular tissue type. These clones are similar in their T-reg phenotype and differ only in the direction of migration to the appropriate tissue which is dictated by unique set of MHC-antigens. T-reg, thymically derived regulatory cell; MHC, major histocompatibility complex; CD, cluster of differentiation. of tumors, thus promoting tolerance and changes to the apoptotic program in tumor tissues (81,88,89). Tolerance may be considered as the deprivation of the informational support from the host to the tumor (90). This data implicates T-regs in the process of tumor progression and the presence of central immune mechanisms that govern tumor spread (2,3). In spite of the fact that the tumor promoting role of tumor-infiltrating immune cells at each step of the metastatic cascade has been stated, interactions between tumor and immune cells are traditionally considered in the context of tolerance or suppression by the immune system only at the tissue level without the evaluation of central, i.e. thymic, regulation of immune cell behavior (2,3,88). Previous studies investigating malignant tissue revealed central mechanisms of immune regulation that define the character, direction and outcomes of cellular interactions at the tissue level (2,48). As this novel therapeutic strategy with checkpoint inhibitors has demonstrated, a functional blockade of the lymphocytes regulating homeostasis in tumor tissue leads to tumor regression due to the deprivation of immunological support from the host (86,90). Therefore, there is a requirement for selective therapeutic agents that target the population of tumor-associated T-regs and not other T-regs in the body.

Conclusion
The current review summarizes the data on the participation of the immune system in the process of tumor growth and metastatic spread. Previous studies have demonstrated the importance of the role of recirculating and tissue-residing immunocompetent cells in tumor progression. However, even a detailed phenotypic specification of lymphoid cells and analysis of their interactions does not completely describe the tumor spread regulation without considering the central part of T-cell competency formation. Central regulation mechanisms provide insight into the predestination and direction of immune reactions in tumor tissue. We believe that enough data pertaining to the regulatory levels of tumor metastasis is available to attempt to influence the process of tumor spread globally, by changing the speed and direction through therapies that target not only tumor cells, but also the regulatory mechanisms of the immune system. For example, the targeted impact on the adaptive immune response with immune checkpoint inhibitors is a successful strategy in immune system interventions in patients with cancer. Finally, elucidating the mechanisms of tumor metastasis with regard to immunological regulation may aid the identification and development of novel therapeutic strategies. A limitation of the present review is that it is purely narrative. Insight into the process of tumor spread through the prism of central immune regulation may be of interest to clinicians, and an in-depth explanation of the mechanisms underlying the efficiency and toxicity of novel immune therapies with checkpoint inhibitors is a topic for future research.


Activity of novel insecticides against different life stages of whitefly ( Bemisia tabaci )

Whitefly, Bemisia tabaci (Gennadius), in North Cotton Growing Zone of India adversely affect cotton yield. Both the nymphal and adult stages of whitefly cause damage by sucking the sap from leaf tissue and honey dew secretion. In this study, the efficacy of new generation insecticides, viz. pyriproxyfen 10EC@1250 ml/ha, spiromesifen 22.9SC @500 ml/ha, flonicamid 50WG @200g/ha, and diafenthiuron 50WP @ 500g/ha were evaluated against different stages of whitefly under confined as well as field conditions in Bt cotton.The experiment conducted in screen house revealed significantly higher per cent reduction of whitefly nymphs in spiromesifen 22.9SC (49.36, 75.17) followed by pyriproxyfen 10EC (33.26, 75.07) in comparison to all other treatments after 5 and 7 days of spray, respectively. Under field conditions, pooled analysis of six experiments showed that flonicamid 50WP was more effective, being at par with pyriproxyfen 10EC, diafenthiuron 50WP, spiromesifen 22.9SC after 10 days of spray as compared to standard check, ethion 50EC and control against whitefly adults. It is concluded that pyriproxyfen10EC and spiromesifen 22.9SC have good nymphicidal action while flonicamid 50WP and diafenthiuron 50WP have adulticidal action.Therefore, for its effective management, these novel insecticides can be rotated alternatively depending upon the stage of pest dominating in the cotton field.

Cotton crop known as white gold is cultivated on an area of 118.77 lakh ha with a production of 484 kg lint/ha in India (Anonymous 2017). After the introduction of Bt cotton, bollworm incidence decreased, however, sucking pest incidence increased. In Punjab, whitefly, B. tabaci has assumed the status of a serious pest of cotton in the recent past and during kharif 2015, it appeared in epidemic form in Punjab and Haryana. Due to the attack of whitefly, the crop productivity of Punjab state fell substantially from 574 kg/ha of lint in 2014-15 to 197 kg/ha in 2015-16 (Dhillon and Sidhu 2016).
Use of insecticides to manage insect pests always remained as preferred tactics. Many new generation insecticides are being used worldwide which are comparatively safer to environment and continuous efforts are needed to evaluate the new eco-friendly insecticides against this pest. The pyriproxyfen and flonicamid are among the new chemistry with novel mode of action. Pyriproxyfen (4-phenoxyphenyl (RS)-2-(2-pyridyloxy) propyl ether) is a potent juvenile hormone analog that acts by suppressing embryogenesis, metamorphosis and inhibit adult formation. It has been found to be effective against variety of insect pests including whiteflies (Qureshi et al. 2009), aphids (Liu and Chen 2001). F l o n i c a m i d ( I K 1 2 2 0 ; N -c y a n o m e t h y l -4trifluoromethylnicotinamide) is a selective insecticide which belongs to the pyridine carboxamide group (Hancock 2003. It inhibits feeding behaviour of aphid and other sucking insect pests (Morita et al. 2014). Spriromesifen, a spirocyclic tetronic acids, acts by inhibiting the lipid synthesis, was found effective against whiteflies (Bretschneider et al. 2003, Nauen and Konanz 2005. Diafenthiuron is another potent insecticide which inhibits the oxidative phosphorylation and disrupts the ATP formation. Diafenthiuron and flonicamid were also found effective for management of jassid and whitefly in Bt cotton (Kalyan et al. 2017). Some novel insecticides have shown specific activity against different life stages of insect pests. Keeping in view the above facts, new generation insecticides, viz. pyriproxyfen, flonicamid, diafenthiuron and spiromesifen having novel mode of action were evaluated against nymphs and adults of whitefly on Bt cotton. The experiment on effect of novel insecticides on nymphal population of whitefly was conducted in screen house at Ludhiana, during kharif 2016. The field efficacy was evaluated at experimental area of PAU Regional Station at Bathinda and Abohar.

MATERIALS AND METHODS
Under screen house conditions: The whitefly culture was maintained on cotton plants in screen house at Entomological Research Farm, PAU, Ludhiana. The leaf cages were prepared from 50 ml centrifuge tubes (3.1 cm diameter × 2.5 cm length). Six circular holes (0.75 cm diameter) were made on sides of tube at equal distance from each other. On the cutting edge of the tube, a fine transparent sheet was glued with fevicol so that the side holes are not covered by this sheet. Muslin cloth was pasted on five holes while the sixth hole was kept uncovered for releasing the whitefly adults into the cage, which was later plugged with a cotton swab. The other end of the tube was fastened on the lower surface of the leaf which was supported from the upper side of leaf by means of a foam piece (10 mm thickness) measuring 3.5 cm × 3.5 cm, pasted on a thin sunmica piece. These leaf cages were attached to the lower surface of cotton leaves and 10 pairs of whitefly were released in the leaf cages. After 24 h, the whiteflies were removed from leaf cages and the number of eggs laid was observed under stereo zoom microscope (40X). In each cage, 50 eggs were retained and other was removed using camel hair brush. The egg development was observed daily. At second or third instar nymphs, the insecticides selected for testing were sprayed on the plants using knapsack sprayer with three replicates. The nymphal population was recorded 5, 7 and 10 days after spray. The per cent reduction of whitefly nymphs over control was calculated.
Under field conditions: The test insecticides were evaluated for their efficacy against whitefly nymphs in field conditions at PAU Regional Station, Bathinda and Abohar. The whitefly nymphal population was recorded in 150 m 2 plot area before spray of insecticides. There are four replications for each treatment. The insecticides were sprayed in each plot in completely randomized block design. The nymphal population was observed in a unit area of leaf (1 cm 2 ) after 3, 7 and 10 days of spray from 3 leaves of 10 randomly selected plant from each plot.
Effect of novel insecticides on adult whitefly population under field conditions: Six experiments were conducted, viz. one at Bathinda and five in Abohar to test efficacy of insecticides against adults of whitefly. The Bt cotton hybrid (RCH773BGII) was grown in the field at each location following recommendations of PAU Package of Practices (Anonymous 2016). The plot size for each treatment was 150 m 2 with four replications in randomised block design.
The insecticides at selected dosages were sprayed using knapsack sprayer, using 375 l of water/ha. The population of whitefly adults was recorded before spray and after 3, 7 and 10 days of insecticide application. The data on number of whitefly adults per three fully opened leaves from upper canopy were recorded from 10 randomly selected plants per replication. The seed cotton yield was also recorded on whole plant basis.
Data Analysis: The data were subjected to ANOVA using arc sine transformation for per cent mortality and square root transformation for population count. The data was analysed using Completely Randomised Block (CRD) and Randomized Block Design (RBD) for screen house and field experiments, respectively. The treatment means were compared using least significant difference (LSD) at 5% probability.

Effect of novel insecticides on whitefly nymphal population
Under screen house conditions: Number of whitefly nymphs did not differ significantly under the leaf cage before spray ( Table 1). The population varied from 49.05-55.97 nymphs in each leaf cage. After 5 day of spray, per cent reduction of whitefly nymphs over control was significantly higher in spiromesifen 22.9SC @500 ml/ha (49.36) followed by pyriproxyfen 10EC @1250 ml/ha (33.26), diafenthiuron 50WP (28.10) and flonicamid 50WG (27.71) which were statistically at par with each other. After 7 days of spray, per cent reduction of whitefly nymphs over control was significantly higher in spiromesifen 22.9SC @500 ml/ha (75.17), being at par with pyriproxyfen 10EC @1250 ml/ha (75.07) followed by diafenthiuron 50WP (61.39). After10 days of spray, it was significantly higher in pyriproxyfen  Under field conditions: At Abohar, after 3 days of spray, per cent reduction of whitefly nymphs was significantly higher in spiromesifen 22.9 SC (24.20) followed by pyriproxyfen 10EC (14.07). Similarly, it was significantly higher in spiromesifen 22.9 SC (67.63) followed by pyriproxyfen 10EC (56.27) after 7 days of spray. However, after 10 DAS, it was significantly higher in pyriproxyfen 10EC (70.60) being at par with spiromesifen 22.9 SC (68.03) ( Table 2).

Control
At Bathinda, after 3 days of spray, non-significant differences were recorded in all test insecticides (Table 2). Whitefly nymphal population reduction was significantly higher in pyriproxyfen 10EC (54.21%) as compared to all other treatments after 7 days of spray. It was followed by spiromesifen 22.9SC (42.35%), diafnethiuron 50WP (33.20%) and flonicamid 50WG (27.03%). However, after 10 days of spray, the maximum reduction of nymphs was recorded in pyriproxyfen 10EC (71.50%) being at par with spiromesifen 22.9 SC (64.26%). Daifenthuiuron 50WP (41.77%) and flonicamid 50 WG (30.21%) were found to be least effective against nymphal population and were statistically at par with each other.
Abohar: At all five experimental locations in Abohar, no significant differences were observed before treatment (Table 4). However, after 3 days of spray, the lowest population was recorded in flonicamid 50WG at all the five locations. The population was again minimum in flonicamid 50WG treatment after 7 days of spray except location IV. Similar, results were observed after 10 days of spray except in experiment no 5. In all experiments, the whitefly adult population was significantly higher in control. The flonicamid 50 WG was significantly at par with pyriproxyfen 10EC after 10 days of spray except in location I.
Seed cotton yield: The pooled seed cotton yield data of Bathinda and two location at Abohar was significantly higher in pyriproxyfen 10EC (24.51 q/ha), flonicamid 50WG (24.52 q/ha) and diafenthiuron 50WP (24.53 q/ha), being at par with each other as compared to all other treatments. However, all the treatments were better than control.
The results showed that new generation insecticides are quite effective in management of whitefly. Among these, flonicamid 50WG and diafenthiuron 50WP were found more effective against adults of whitefly after 3, 7 and 10 days of spay. However, these did not show significant decrease in nymphal population. In contrast to these, pyriproxyfen 10EC and spiromesifen 22.9SC proved more effective against nymphs of whitefly, while these insecticides lowered the adult whitefly population after 10 days of spray. Hence, it is imperative from the data that pyriproxyfen 10EC and spiromesifen 22.9SC has good nymphicidal and flonicamid 50WP and diafenthiuron 50WP has adulticidal action. It has been reported that pyriproxyfen does not possess direct toxicity against whitefly adults, however, it exhibits juvenoidal activity  by disrupting normal hormonal balance, resulting in the suppression of embryogenesis, metamorphosis and adult formation (Ishaaya and Horowitz 1995, Kontsedalov et al. 2009, Roditalis et al. 2014. Earlier researchers also reported that flonicamid was effective against whitefly (Kontsedalov et al. 2009, Roditalis et al. 2014) and leaf hoppers (Kodandaram et al. 2017) in okra. Diafenthiuron and flonicamid were also reported to be effective against whitefly in Bt cotton (Kalyan et al 2017). Treatment with spiromesifen @5 mg/l caused 40% whitefly adult mortality and reduced the fecundity by more than 80% (Kontsedalov et al. 2009). Further number of ovarioles in female was also significantly reduced. Therefore, in integrated pest management programme, these novel insecticides can be rotated alternatively depending upon the stage of whitefly dominating in the fields.


extfieldsofstudy: Chemistry Medicine

text:
The Impairments of α-Synuclein and Mechanistic Target of Rapamycin in Rotenone-Induced SH-SY5Y Cells and Mice Model of Parkinson’s Disease

Parkinson’s disease (PD) is characterized by selective degeneration of dopaminergic (DAergic) neurons in the substantia nigra pars compacta (SNpc). α-synuclein (α-syn) is known to regulate mitochondrial function and both PINK1 and Parkin have been shown to eliminate damaged mitochondria in PD. Mechanistic target of rapamycin (mTOR) is expressed in several distinct subcellular compartments and mediates the effects of nutrients, growth factors, and stress on cell growth. However, the contributions of these various regulators to DAergic cell death have been demonstrated mainly in culture with serum, which is known to dramatically influence endogenous growth rate and toxin susceptibility through nutrient and growth factor signaling. Therefore, we compared neurotoxicity induced by the mitochondrial inhibitor rotenone (ROT, 5 or 10 μM for 24 h) in SH-SY5Y cells cultured with 10% fetal bovine serum (FBS), 1% FBS, or 1% bovine serum albumin (BSA, serum-free). In addition, C57BL/6J mice were injected with 12 μg ROT into the right striatum, and brains examined by histology and Western blotting 2 weeks later for evidence of DAergic cell death and the underlying signaling mechanisms. ROT dose-dependently reduced SH-SY5Y cell viability in all serum groups without a significant effect of serum concentration. ROT injection also significantly reduced immunoreactivity for the DAergic cell marker tyrosine hydroxylase (TH) in both the mouse striatum and SNpc. Western blotting revealed that ROT inhibited TH and Parkin expression while increasing α-syn and PINK1 expression in both SH-SY5Y cells and injected mice, consistent with disruption of mitochondrial function. Moreover, expression levels of the mTOR signaling pathway components mTORC, AMP-activated protein kinase (AMPK), ULK1, and ATG13 were altered in ROT-induced PD. Further, serum level influenced mTOR signaling in the absence of ROT and the changes in response to ROT. Signs of endoplasmic reticulum (ER) stress and altered expression of tethering proteins mediating mitochondria-associated ER contacts (MAMs) were also altered concomitant with ROT-induced neurodegeneration. Taken together, this study demonstrates that complex mechanism involving mitochondrial dysfunction, altered mTOR nutrient-sensing pathways, ER stress, and disrupted MAM protein dynamics are involved in DAergic neurodegeneration in response to ROT.

INTRODUCTION
Parkinson's disease (PD) is an age-related neurodegenerative disorder (NDD) characterized by progressive loss of dopaminergic (DAergic) neurons in the substantia nigra (SN) pars compacta (SNpc) along with intracellular aggregation of α-synuclein (α-syn) in structures known as Lewy bodies (LBs) (Lee and Trojanowski, 2006). Neurons use multiple feedback controls to regulate metabolism in response to nutrients and other signals. Neurons depend on oxidative phosphorylation (OXPHOS) for most energy needs, a process that consumes oxygen and glucose to generate energy-storing ATP (Hsu and Sabatini, 2008). This process relies on electron flow via electron transport chain (ETC) components in the inner mitochondrial membrane, culminating in the reduction of oxygen in the matrix, and the generation of a membrane potential across the inner membrane that is exploited to convert ADP to ATP (Whitworth and Pallanck, 2017).
Rotenone (ROT) is a naturally occurring insecticide, pesticide, and piscicide extracted from the roots of plants of the genera Lonchocarpus and Derris. It is highly lipophilic and therefore easily crosses all biological membranes including blood-brain barrier (Martinez and Greenamyre, 2012). The mitochondrial toxin ROT is widely used to induced a PDlike pathology in culture cells and experimental animals. ROT impairs OXPHOS by inhibiting mitochondrial ETC complex I (reduced nicotinamide adenine dinucleotideubiquinone reductase), leading to reduced ATP production and the formation of reactive oxygen species (ROS) that can induce oxidative stress (Duty and Jenner, 2011;Martinez and Greenamyre, 2012). The major advantages of ROT treatment for PD modeling are its ability to induce α-syn-positive cytoplasmic inclusions in nigral neurons resembling LBs (Betarbet et al., 2000) and progressive neurodegeneration accompanied by PD-like motor and non-motor symptoms (Johnson and Bobrovskaya, 2015).
In addition to external stressors, nutrient signals, growth factors, and cell energy balance (a product of mitochondrial function) are major regulators of cellular growth, proliferation, and survival in health and disease (Yang et al., 2019). However, the signaling pathways underlying nutrient effects in PD are largely unknown. Mechanistic/mammalian target of rapamycin (mTOR) is a serine-threonine kinase that controls several important aspects of mammalian cellular function through nutrient signal transduction (Saxton and Sabatini, 2017). It exists in two distinct multiprotein complexes, mTORC1 and mTORC2 (Switon et al., 2017), each with its own unique subunit composition and functions. The mTORC1 complex comprised of mTOR, mLST-8, FKBP38, Deptor, PRAS40, and rapamycin-sensitive adaptor protein of mTOR (Raptor) regulates cell growth, proliferation, and metabolism (Bai and Jiang, 2010), whereas mTORC2 comprised of mTOR, mSIN1, mLST8, and the rapamycin-insensitive subunit Rictor (Laplante and Sabatini, 2009) controls cell survival and cell-cycle dependent cytoskeleton assembly. Each complex also utilizes distinct cofactors and substrates for regulation of these processes according to nutrient and energy status. Collectively, these two complexes regulate multiple physiologic processes (Morrison Joly et al., 2016), such as axonal growth, neuronal development and survival, and synaptic plasticity, thereby contributing to learning and memory (Jaworski and Sheng, 2006;Swiech et al., 2008). The AMP-activated protein kinase (AMPK) is another widely recognized energy-sensing serine/threonine kinase (Mihaylova and Shaw, 2011). AMPK responds to oxidative stress and critically involved in NDDs (Jiang et al., 2013). In addition, AMPK-driven mTOR downregulation serves as a turn-off switch of the cellular anabolic program (Swiech et al., 2008). AMPK directly regulates the autophagy-associated kinase ULK1 through phosphorylation under nutrient signaling. Moreover, mTORC1 also phosphorylates ULK1 at Ser757 and affect the interaction between ULK1 and AMPK Shang et al., 2011). Therefore, these mutually interacting protein signaling pathways may sense and integrate countless stimuli from nutrients and growth factors to direct normal cellular processes and pathogenic processes in NDDs like PD (Linke et al., 2017).
Despite extensive research efforts, it is still largely unclear how nutrients regulate the protein signaling pathways relevant to NDDs such as PD. Fetal bovine serum (FBS) has been used for mammalian cell culture to promote growth, differentiation, and survival (Piletz et al., 2018). However, serum is not a physiological fluid in vivo, and has been shown to induce aberrant cell growth characteristics, alter phenotype, and suppress neurotoxicity in vitro (Pirkmajer and Chibalin, 2011;Tekkatte et al., 2011). As many of these investigations have employed cells cultured in serum, the pathogenic pathways underlying the ROT-induced PD phenotype may differ substantially from PD pathogenesis in vivo. Therefore, it is critical to examine the effects of serumcontaining cell culture media on cellular models of PD. Here we examined ROT toxicity in SH-SY5Y neuroblastoma cells under three different cell culture conditions: (1) 10% FBS, (2) low (1%) FBS, and (3) serum-free medium (0% FBS) containing 1% bovine serum albumin (BSA) [used as synthetic serum as previously reported (Ramalingam and Kim, 2016)]. We also compared results to stereotaxic ROT injection in C57BL/6J mice. This study aimed to elucidate the molecular mechanism underlying ROT-induced toxicity, specifically the distinct contributions of mTOR/AMPK, and ER-mitochondrial tethering pathways.

Cell Culture and Treatment
The human neuroblastoma cell line SH-SY5Y (CRL-2266) was obtained from ATCC (Manassas, VA, United States) and maintained in DMEM supplemented with 10% FBS, Pen Strep (100 U/ml; 100 mg/ml), and 2mM L-glutamine, at 37 • C in a humidified atmosphere containing 5% CO 2 /95% air. Confluent cultures were washed with phosphate-buffered saline (PBS), detached with 0.25% trypsin-EDTA solution, reseeded as 1 × 10 5 cells/ml of DMEM containing 10% FBS or 1% FBS or 1% BSA and used for experiments after overnight incubation. SH-SY5Y cells were incubated with the absence or presence of ROT for 24 h. Combining floating cells in the medium and adherent cells detached by trypsinization and subjected to cell counting and Western blotting.

Cell Counting and Cell Morphology
After treated with ROT or solvent control (DMSO) at the indicated concentrations for 24 h, phase contrast images were taken using microscope Olympus CKX41 equipped with a camera. Damaged and deplated floating cells in the medium and adherent cells detached by trypsinization were combined and subjected to trypan blue cell counting method. Surviving cells, which cannot be stained with trypan blue dye, were counted under microscope. The cell count assay was performed in triplicates and expressed as a percentage (%) of control.

Preparation of Total Cell Lysates and Immunoblotting
After treated with indicated concentrations of ROT or DMSO for 24 h, cells were harvested by scraping with media, pelleted and washed twice with PBS. Then, exposed to RIPA buffer (25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% Nonidet P-40, 0.251% sodium deoxycholate, 1% sodium dodecyl sulfate (SDS): Thermo Fisher Scientific, United States) supplemented with protease and phosphatase inhibitors cocktail (Thermo Fisher Scientific, United States) and incubated for 30 min in ice. Lysates were centrifuged at 13,000 rpm for 20 min at 4 • C and the supernatants were collected as total cell lysate. Protein concentrations were determined by BCA method (Kit). Proteins (30 µg) were separated on 6-12% SDS-polyacrylamide gels and transferred to PVDF membranes (Millipore, Bellerica, MA, United States). The membranes were washed with Tris buffered saline (TBS; 10 mM Tris-HCl, 150 mM NaCl, pH 7.5) containing 0.5% (v/v) Tween 20 (TBST) followed by blocking with 5% (v/v) non-fat dried milk solution prepared in TBST and then incubated overnight with primary antibodies at 4 • C. The antibodies used are listed in Supplementary Table 1. After this, membranes were exposed to secondary antibodies conjugated to horseradish peroxidase for 2∼3 h at room temperature and further washed thrice with TBST. The immunoreactivity was detected by the luminolbased chemiluminescence (ECL) system. Equal protein loading was assessed by the expression level of β-actin. Densitometric analysis was performed using ImageJ (National Institute of Health, Bethesda, MD, United States) software.

Triton-X-100-Soluble and -Insoluble Fractionation
Following ROT toxicity for 24 h, SH-SY5Y cells were lysed on ice in RIPA buffer containing protease and phosphatase inhibitors with 1% Triton-X-100 for 30 min. Lysates were centrifuged at 12,000 rpm for 20 min at 4 • C and the supernatants were collected as Triton-X-100-souble fraction. The cell pellets were washed with PBS then dissolved in the RIPA buffer containing protease and phosphatase inhibitors with 1% Triton-X-100 and 2% SDS and sonicated for 10 s and used as Triton-X-100-insoluble fraction. Protein samples were immunoblotted as described above.

Animals and Stereotaxic Surgery
Five-week-old C57BL/6J male mice were purchased from DBL (South Korea) were housed at room temperature under 12 h ligh/dark cycle. Food and water were provided ad libitum for 1 week before intrastriatal surgery. All animal experiments were approved by the Ethical Committee of Animal Research of DGIST, Daegu, South Korea accordance with international guidelines (DGIST-IACUC-18010204-01). Mice underwent unilateral stereotaxic surgery under injectable 2.5% Avertin anesthesia. A hole was drilled in the skull and a cannula inserted at following stereotaxic coordinates at AP + 1.0, ML −2.5 from bregma and DV −3.0 below dura in the right striatum, and 12 µg of freshly prepared ROT (dissolved in 2 µl DMSO) was infused (0.2 µl/min for 10 min for infusion with 5 min for diffusion). Control animals were injected with vehicle DMSO. Fourteen days after surgery, mice were anesthetized with avertin and perfused.

Immunohistochemistry
Mice were sacrificed by terminal anesthesia and transcardially perfused with 50 ml PBS followed by 50 ml 4% paraformaldehyde (PFA). Brains were rapidly removed, post-fixed in 4% PFA for 24 h and stored in a 30% sucrose solution for 48 h or more. Serial coronal sections (35 µm) were cut using a freezing sledge microtome and a 1:4 series of sections was used for all quantitative immunohistochemistry. For immunohistochemical analyses, blocking of non-specific secondary antibody binding (using 3% normal horse serum in PBS with 0.2% Triton X-100 at room temperature for 1 h), sections were incubated overnight at room temperature with the primary antibody of TH (1:1000) diluted in PBS with 0.2% Triton X-100. Sections were then incubated in a biotinylated secondary antibody for 1 h [horse anti-mouse (1:2000, Vector, United Kingdom)] followed by 1 h incubation in streptavidin-biotin-horseradish peroxidase solution (Vector, United Kingdom). Sections were developed in 0.5% solution of diaminobenzidine (DAB) tetrahydrochloride (Sigma, Ireland) and mounted on microscope slides coverslipped using DPX mountant (BDH chemicals, United Kingdom). Immunostained sections were photographed and the total number of TH positive neurons in the SNpc was determined using the Optical fractionator probe in Stereo Investigator software (MicroBrightfield, Williston, VT, United States). All stereological counting was performed in a blinded manner to mice treatments.

Cell Lysates Preparation and Immunoblotting
After fourteen days of surgery, mice were anesthetized with avertin and perfused with PBS. Mice brain subregions of midbrain (MB) and striatum (ST) were located following procedures described previously (Jackson- Lewis and Przedborski, 2007). The lysates were prepared and Western blotting done as mentioned above.

Statistical Analysis
Data are expressed as mean ± standard error mean (SEM). The significance level of treatment effects was determined using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test (in vitro; three or more groups) or an paired/unpaired two-tailed Student's t-test (in vivo; two-group comparisons). A probability of <5% (p < 0.05) was considered to be statistically significant. GraphPad Prism 5.0 software (La Jolla, CA) was used for data analyses and preparation of all graphs.

Rotenone-Induced Death in SH-SY5Y Cells
SH-SY5Y cells were cultured in the same medium containing different serum concentrations and then treated with ROT (0, 0.5, 1, 2.5, 5, and 10 µM) for 24 h. Microscopic examination revealed that the majority of cells were damaged and deplated following 5 and 10 µM ROT treatment in 1% FBS and 1% BSA culture medium (Supplementary Figure 1). Trypan blue cell viability assays combining both floating and adherent cells detached by trypsinization revealed little proliferation in 1% FBS or 1% BSA (p < 0.001) compared to control 10% FBS culture media. ROT dose-dependently increased cell death in all groups after 24 h (all p < 0.001; Figure 1A). However, cell death was substantially greater in the low-serum and no-serum groups.
FIGURE 1 | SH-SY5Y cells were seeded as 1 × 10 5 cells/ml of DMEM containing 10% FBS or 1% FBS or 1% BSA and used for experiments after overnight incubation. Cells were incubated with the absence or presence of different concentrations of ROT (0, 0.5, 1, 2.5, 5, and 10 µM) for 24 h and assessed for trypan blue assay (A) or TH, PINK1, Parkin, α-syn and β-actin by Western blotting (B). In addition, cells were fractionated into 1% Triton X-100 soluble and insoluble fractions and analyzed for α-syn by Western blotting (C). Each picture is a representative of three independent experiments. Data are mean ± SEM of three independent experiments and analyzed by one-way of variance (ANOVA) followed by Tukey's post hoc test. Statistical significance: a compared with 10% FBS control; b compared with 1% FBS control; c compared with 1% BSA control; * p < 0.05, * * p < 0.01, and * * * p < 0.001.
Parkinson's disease is characterized by the presence of abnormal intracellular α-syn inclusions. ROT treatment increased α-syn in total cell lysates from all three serum concentrations groups (p < 0.001; Figure 1B), suggesting that ROT reduces SH-SY5Y DAergic neuron viability by promoting α-syn accumulation. Separate Western blot analyzes of the Triton-X100-soluble and insoluble lysate fractions, which are thought to include bioavailable and aggregated α-syn proteins, respectively revealed an increase of the oligomeric form in the Triton-X100-insoluble fraction and an increase of the monomeric form in the Triton-X100-soluble fraction. Thus, ROT may induce SH-SY5Y cell death through enhanced α-syn production and ensuing aggregation ( Figure 1C).

Rotenone Alters mTORC and AMPK Expression Levels in SH-SY5Y Cells
The activities of mTORC1 and mTORC2 were assessed by measuring expression levels of phosphorylated (p)-mTORC1 (Ser2448) and p-mTORC2 (Ser2481), respectively. Control cells cultured in 1% FBS or 1% BSA exhibited slightly increased p-mTORC1 and significantly decreased p-mTORC2 (p < 0.001 and p < 0.01, respectively) (Figure 2A) compared to control cells cultured in 10% FBS. ROT (10 µM) dramatically increased p-mTORC1 (p < 0.05) and decreased p-mTORC2 (p < 0.001) expression in cells cultured with 10% FBS. Conversely, ROT decreased p-mTORC1 (p < 0.05 and p < 0.001, respectively) and increased p-mTORC2 (both p < 0.001) in cells cultured with 1% FBS or 1% BSA. These data suggest that FBS levels and ROT both alter mTOR signaling pathways in SH-SY5Y cells and that the effects of ROT differ depending on serum levels (nutrient availability). The overall effect of mTOR signaling depends on the specific complex activated, mTORC1 or mTORC2, which are distinguished by Raptor in mTORC1 and Rictor in mTORC2. Thus, we measured expression levels of p-Raptor (Ser792) and p-Rictor (Thr1135). Control cells cultured in 1% FBS or 1% BSA demonstrated no difference in p-Raptor expression but increased p-Rictor expression (p < 0.01 and p < 0.001, respectively) compared to control SH-SY5Y cells cultured in 10% FBS (Figure 2B), suggesting mTORC2 signaling predominance under low nutrient conditions. ROT treatment (10 µM for 24 h) decreased the expression levels of both p-Raptor (both p < 0.001) and p-Rictor (p < 0.05 in 10% and 1% FBS; p < 0.01 in BSA) in all three culture conditions, while total mTOR, Raptor and Rictor expression levels were unaffected (Figures 2A,B).
AMP-activated protein kinase is a major metabolic energy sensor that contributes to mTOR signaling through interactions with ULK1 and ATG13. Control cells cultured in 1% FBS or 1% BSA showed increased expression levels of p-AMPK (Thr172) (both p < 0.001), p-ULK1 (Ser757) (p < 0.001 and p < 0.05), and ATG13 (both p < 0.001) compared to control cells cultured in 10% FBS (Figure 2C). Treatment with ROT (10 µM for 24 h) significantly enhanced p-AMPK (Thr172), p-ULK1 (Ser757), and ATG13 expression by SH-SY5Y cells cultured in 10% FBS (all p < 0.001) but decreased expression levels of all three phosphorylated proteins in SH-SY5Y cells cultured with 1% FBS and 1% BSA, again indicating that serum influences mTOR signaling independently of ROT, and alters the mTOR signaling change in response to ROT.

Rotenone Induces ER Stress and Disrupts MAM in SH-SY5Y Cells
The endoplasmic reticulum is the central organelle responsible for protein folding, and there is compelling evidence that ER stress and protein misfolding are involved in ROT-induced PD-like toxicity. To examine ER stress in SH-SY5Y cells, we measured changes in the expression levels of protein kinase RNA (PKR)-like ER kinase (PERK) and inositol-requiring enzyme 1 α (IRE-1α). ROT (10 µM) increased expression of p-PERK at Thr981 (p < 0.001) and IRE-1α (p < 0.01) (Figure 3), suggesting that ER stress is involved in ROT-induced neuronal dysfunction. We further investigated the protein expression levels of MAM tethering proteins GRP75, Mfn1, and Mfn2. Control cells cultured with 1% FBS or 1% BSA showed significantly increased expression levels of GRP75 (both p < 0.001), Mfn1 (p < 0.01 in 1% FBS and p < 0.001 in 1% BSA), and Mfn2 (both p < 0.05) compared to control cells in 10% FBS (Figure 3). In addition, ROT (10 µM for 24 h) further increased the protein expression levels of GRP75 (p < 0.001) in all culture conditions. However, Mfn1 and Mfn2 were significantly increased by ROT (10 µM for 24 h) in cells cultured with 10% FBS (p < 0.01 and p < 0.05, respectively) but decreased in cells cultured in 1% FBS (both p < 0.05) or 1% BSA (p < 0.001 for Mfn1 and p < 0.01 for Mfn2).

Intrastriatal Rotenone Injection Alters TH, PINK1, Parkin, and α-Syn Expression Levels in C57BL/6J Mice
To examine the effects of ROT on mTOR signaling, ER stress, mitochondrial function, MAM function, and cell viability in vivo, we conducted single unilateral intrastriatal ROT infusions (12 µg) in C57BL/6J mice. Instrastriatal injection induced, significant depletion of TH immunoreactivity in the striatum and SN (Figure 4A), and significantly reduced TH-positive cell numbers in the ipsilateral (injection-side) SNpc after 14 days (p < 0.01). The expression levels of TH and Parkin were decreased while PINK1 and α-syn expression levels were increased in midbrain ( Figure 4B) and striatum ( Figure 4C) of ROT-injected mice compared to vehicle (DMSO)-injected mice FIGURE 2 | SH-SY5Y cells were seeded as 1 × 10 5 cells/ml of DMEM containing 10% FBS or 1% FBS or 1% BSA and used for experiments after overnight incubation. Cells were incubated with the absence or presence of ROT (0, 5, and/or 10 µM) for 24 h and assessed for mTOR (A,B) and AMPK (C) signaling proteins and β-actin by Western blotting. Each picture is a representative of three independent experiments. Data are mean ± SEM of three independent experiments and analyzed by one-way of variance (ANOVA) followed by Tukey's post hoc test. Statistical significance: a compared with 10% FBS control; b compared with 1% FBS control; c compared with 1% BSA control; * p < 0.05, * * p < 0.01, and * * * p < 0.001. FIGURE 3 | SH-SY5Y cells were seeded as 1 × 10 5 cells/ml of DMEM containing 10% FBS or 1% FBS or 1% BSA and used for experiments after overnight incubation. Cells were incubated with the absence or presence of ROT (0, 5, and 10 µM) for 24 h and assessed for ER and MAM tethering proteins and β-actin by Western blotting. Each picture is a representative of three independent experiments. Data are mean ± SEM of three independent experiments and analyzed by one-way of variance (ANOVA) followed by Tukey's post hoc test. Statistical significance: a compared with 10% FBS control; b compared with 1% FBS control; c compared with 1% BSA control; * p < 0.05, * * p < 0.01, and * * * p < 0.001. as measured by Western blotting, consistent with the effects of SH-SY5Y cells.

Intrastriatal Rotenone Alters mTOR and AMPK Signaling in C57BL/6J Mice
We then investigated the involvement of mTOR pathways in ROT toxicity. ROT injection increased p-mTORC1 (Ser2448) expression (p < 0.01) but reduced p-mTORC2 (Ser2481) expression (p < 0.05) in mouse midbrain ( Figure 5A). Alternatively, ROT dramatically decreased both p-mTORC1 and p-mTORC2 in mouse striatum (p < 0.001; Figure 5C). In addition, p-Raptor and p-Rictor expression levels were reduced in the midbrain of ROT-injected mice (p < 0.01 and p < 0.001, respectively) ( Figure 5A). Collectively, these findings suggest that ROT has region-specific effects on mTOR signaling pathways. Moreover, the protein expression levels of p-AMPK, p-ULK1, and ATG13 were decreased in ROT-injected mice (all p < 0.01), while t-AMPK and t-ULK1 expression levels remained unchanged (Figure 5B). In the striatum of ROT-injected mice, p-AMPK was increased while p-ULK1 and ATG13 expression levels were reduced ( Figure 5D).

Intrastriatal Rotenone Alters ER Stress and Disrupts MAM in C57BL/6J Mice
To examine possible ROT-induced ER stress in the brain, we measured expression of the ER stress-associated proteins PERK and IRE-1α in midbrain ( Figure 6A) and striatum (Figure 6B) lysates. Expression levels of p-PERK (Thr981) and IRE-1α were markedly increased by ROT in both midbrain and striatum (p-PERK: p < 0.001 in both regions; IRE-1α: p < 0.001 in midbrain, p < 0.01 in striatum) implicating ER stress in ROT induced neurodegeneration. Moreover, ER malfunction involved MAM dysfunction as the MAM tethering proteins GRP75, Mfn1, and Mfn2 were downregulated in midbrain (p < 0.05 for GRP75 and Mfn1; p < 0.01 for Mfn2) and striatum (p < 0.05 for GRP75; p < 0.001 for Mfn1 and Mfn2) of ROT-injected mice.

DISCUSSION
In recent years, many studies have been conducted to elucidate the molecular pathophysiology of PD progression. ROT, a fish poison that inhibits mitochondrial complex I, induces PD-like changes in cultured neurons, and rodent FIGURE 4 | Photomicrograph of ROT lesioned C57BL/6J mice striatum and substantia nigra pars compacta (SNpc) immunostained for TH (25×). The quantitative image analysis of TH positive cells in SNpc expressed as percentage (%). Each picture is a representative of three independent experiments. Data are mean ± SEM (n = 3) and analyzed by paired two-tailed Student's t-test. * * p < 0.01 (A). Effects of ROT on TH, PINK1, Parkin, α-syn, and β-actin in the midbrain (B) and striatum (C) of stereotaxic C57BL/6J mice were analyzed by Western blotting. Data are mean ± SEM (n = 4 for control; n = 5 for ROT) and analyzed by unpaired two-tailed Student's t-test. * p < 0.05, * * p < 0.01, and * * * p < 0.001.
FIGURE 6 | Effects of ROT on ER and MAM pathways in the midbrain (A) and striatum (B) of stereotaxic C57BL/6J mice were analyzed by Western blotting. Data are mean ± SEM (n = 4 for control; n = 5 for ROT) and analyzed by unpaired two-tailed Student's t-test. * p < 0.05, * * p < 0.01, and * * * p < 0.001.
brain (Sherer et al., 2003). SH-SY5Y is a DA-producing human catecholaminergic neuroblastoma cell line widely used as an in vitro dopaminergic cell model (Martins et al., 2013). ROT treatment for 24 h dose-dependently reduced SH-SY5Y cell number. Moreover, serum starvation by culture in 1% FBS or 1% BSA medium inhibited cell growth and proliferation in the absence of ROT. Cells under serum starvation alone or with ROT detached from the culture surface and lost typical neuronal morphology within 24 h consistent with previous studies showing that serum starvation induces cell death (Chou and Yung, 1997) and that ROT-induced impairment of mitochondrial complex I activity leads to apoptosis, likely via excess ROS formation (Imamura et al., 2006). Tyrosine hydroxylase, the rate-limiting enzyme in DA synthesis, is obviously critical for phenotypic expression (Nagatsu et al., 1964). ROT treatment for 24 h dramatically reduced TH expression in SH-SY5Y cells. Moreover, ROT infusion in mouse striatum reduced TH immunoreactivity and reduced THpositive cell count in the SNpc after 14 days, consistent with the dopaminergic neuronal degeneration along the nigrostriatal pathway that parallels the symptoms of PD (Carriere et al., 2016). In addition, surviving cells accumulate damaged mitochondria, leading to metabolic deficits, oxidative stress, mitophagy, and greater susceptibility to other pathogenic processes such as protein aggregation (Shaltouki et al., 2015).
Parkin (PARK2), a cytosolic ubiquitin ligase, and PTENinduced kinase 1 (PINK1; PARK6), a mitochondria-targeted kinase, act important mediators of mitochondrial quality control (Whitworth and Pallanck, 2017) by removing damaged or dysfunctional mitochondria and preserving a healthy mitochondrial population (Oh et al., 2017). Dysfunctional mitochondria can trigger cell degeneration via mitophagy in PD (Li et al., 2015). In the present study, ROT inhibited Parkin expression and enhanced PINK1 expression in SH-SY5Y cells as well as in mouse midbrain and striatum, suggesting that loss of coordinated Parkin/PINK1 function contributes to ROT-induced mitochondrial impairment, oxidative stress, and cell death. Loss of Parkin results in an initial accumulation of damaged mitochondria while PINK1 accumulation may cause proteosomal dysfunction (Wang et al., 2005;Shaltouki et al., 2015), which reduces Parkin solubility in toxin-induced PD models. Moreover, reduced Parkin in turn leads to the formation of protein aggregates resembling LBs in PD (Um et al., 2009(Um et al., , 2010. Protein aggregation and filament formation are histopathological hallmarks of NDDs. For instance, PD is characterized by abnormal intracellular protein inclusions (LB and Lewy neurites or LN) mainly composed of aggregated α-syn fibrils (Faustini et al., 2017). The aggregation of α-syn causes proteasomal dysfunction, which may lead directly to neurodegeneration (Tanaka et al., 2001). We observed α-syn accumulation in SH-SY5Y cells and mouse midbrain and striatum following ROT treatment, consistent with previous studies demonstrating that ROT can trigger α-syn accumulation both in vitro and in vivo (Yuan et al., 2015). Aggregation of α-syn can be spread via a prion-like mechanism to neighboring neurons (Bae et al., 2012), resulting in the functional decline and death of dopaminergic neurons throughout the SNpc (Li et al., 2008;Lee et al., 2010). Moreover, α-syn can bind to TH, so accumulation of α-syn can reduce DAergic transmission by surviving cells (Perez and Hastings, 2004).
Under normal conditions, α-syn is cleared by proteolytic degradation in the extracellular space or lysosomal degradation in neighboring cells (Sung et al., 2005;Lee et al., 2008). However, impairment of these degradative mechanisms or an incapacity to clear proteins that have already aggregated is proposed the as primary defect leading to accumulation of insoluble α-syn (Cantuti-Castelvetri et al., 2005) within the LBs characteristic of PD (Myohanen et al., 2012), dementia with LB, and multiple system atrophy (Iwata et al., 2003). Previous in vitro studies have reported that the Triton-X100-soluble fraction represents bioavailable α-syn, whereas the Triton-X100-insoluble component may represent α-syn aggregated or sequestered in oligomeric forms (Klucken et al., 2003;Alves da Costa et al., 2006). In our present study, the oligomeric form of α-syn was detected only in the Triton-X100-insoluble fraction, while the monomeric form of α-syn was detected only in the Triton-X100-soluble fraction. ROT increased both Triton-X100-insoluble α-syn oligomers and Triton-X100-soluble monomer, consistent with previous studies reporting that ROT interacts with α-syn to drive accumulation of insoluble forms in SH-SY5Y cells (Sherer et al., 2002;Lee et al., 2004).
Growth factors and nutrients enhance protein synthesis and suppress protein degradation . mTORs integrate signals from nutrients and growth factors with current energy status to regulate many neuronal processes, including autophagy, ribosome biogenesis, and growth (Sarbassov et al., 2005), as well as synaptic plasticity, learning and memory, and food uptake in adult brain (Zhou et al., 2015). Our present study suggests that both mTORC1 and mTORC2 regulate the responses to FBS and ROT in SH-SY5Y cells. ROT increased p-mTORC1 but decreased p-mTORC2 protein expression in the presence of 10% FBS, consistent with changes in the midbrain following ROT injection. Therefore, mTORC1 appears to be activated by ROT in the presence of sufficient nutrients and growth factors. The exact mechanism by which mTORC1 is activated and mTORC2 inhibited by ROT under this nutrient-rich condition requires additional studies. Control cells treated with 1% BSA also exhibited enhanced p-mTORC1 and reduced p-mTORC2 consistent with a previous study (Peruchetti et al., 2014). Conversely, ROT decreased p-mTORC1 and increased p-mTORC2 expression in the presence of 1% FBS or 1% BSA, changes shown to directly inhibit cell growth, and mitochondrial proteins during nutrient starvation. Our results are in line with another study reporting decreased mTORC1 in PC12 cells following ROT treatment for 24 h (Laplante and Sabatini, 2009). These results suggest that ROT treatment under nutrient shortage (serum starvation) inhibits mTORC1, resulting in lower mitochondrial membrane potential, oxygen consumption, and cellular ATP levels (Schieke et al., 2006).
Studies also suggest that mTORC1 acts as a negative regulator of mTORC2 (Dibble et al., 2009;Xie and Proud, 2014). Another study found that Rheb activated mTORC1 but inhibited mTORC2, while TSC1/2 inhibited Rheb/mTORC1 but activated mTORC2, most likely by overcoming the negative feedback loop (Yang et al., 2006). To further explore the role of mTOR complex signaling pathways in ROT toxicity, we examined expression of Raptor, a required cofactor for rapamycin-sensitive mTORC1 signaling, and Rictor, a regulatory subunit of mTORC2 (Li et al., 2007). ROT treatment decreased the phosphorylation levels of Raptor and Rictor both in vitro and in vivo, inconsistent with the aforementioned changes in mTORC1 and mTORC2 activity and suggesting that ROT can differentially stimulate or inhibit mTOR complex activities under different nutrient conditions; however, further investigations using specific substrates of these mTOR complexes necessary to examine the underlying mechanisms. mTORC1 is the major transducer of nutrient signaling for cell growth (Efeyan et al., 2012). Similarly, AMPK senses energy deficiency in the form of an increased AMP/ATP ratio to regulate a myriad of cellular processes (Poels et al., 2009). The substrates ULK1 (ATG1) and ATG13 function downstream of mTORs and AMPK (Hosokawa et al., 2009;Egan et al., 2011). In this study and others (Wu et al., 2011), ROT-induced AMPK phosphorylation at Thr172 in SH-SY5Y cells cultured with 10% FBS medium. Increased p-AMPK was also reported in ROT treated HepG2 cells (Hou et al., 2018). Furthermore, activated ULK1 was shown to directly phosphorylate AMPK (Loffler et al., 2011). In contrast, Sciarretta et al. (2018) reported that ROT decreased mTORC1 and inhibited AMPK-ULK1 under serum starvation. Under starvation, AMPK translocates to the lysosome and both lysosomal AMPK and mTORC1 contribute to autophagy via ULK1/ATG13/FIP200 regulation (Ha et al., 2015). In this study, ROT inhibited mTORC1, AMPK/ULK/ATG13 in cells cultured with 1% FBS or 1% BSA (nutrient starvation). In ROT-injected mouse midbrain, the activation of mTORC1 by ROT inhibited the AMPK/ULK1/ATG13 pathway after 14 days. From the above results, we speculate that the nutrient-sensing molecules mTORC1 and AMPK/ULK1/ATG13 are differently regulated by ROT toxicity in vitro and in vivo.
The endoplasmic reticulum is involved in protein folding, maintenance of Ca 2+ homeostasis and cholesterol synthesis, and ER dysfunction is implicated in the pathogenesis of α-syn mediated NDDs (Paillusson et al., 2017). The ER depends on ATP to correct misfolded protein errors, so ROT-induced ATP reduction can lead to ER stress, which in turn initiates the UPR through activation of PERK, and IRE-1α (Jiang et al., 2016). In the present study, ROT enhanced p-PERK Thr981 and IRE-1α expression levels, indicating ROT-induced ER stress, in both SH-SY5Y cells and mouse midbrain and striatum. This enhanced activation of PERK signaling results in a sustained reduction of global protein synthesis, leading to neuronal loss . In addition, PERK over-activation has been observed in postmortem brain and spinal cord tissues of NDD patients (Smith and Mallucci, 2016). Taken together, the present study suggests that ROT-induced ER stress may trigger the death of SH-SY5Y cells and DAergic neurons in mice.
Mitochondria-associated ER membrane abnormalities have been described in cellular models of a number of NDDs, including PD (Cali et al., 2013). GRP75 is essential for maintaining physical contact between the ER and mitochondria, thereby facilitating Ca 2+ exchange and transfer through ERbound IP 3 R and mitochondrial VDAC1 (Honrath et al., 2017). Expression of GRP75 was increased in SH-SY5Y cells by ROT treatment but decreased in midbrain and striatum of C57BL/6J mice. As previously reported, GRP75 overexpression in human DAergic cells enhanced vulnerability to ROT-induced cytotoxicity (Jin et al., 2006), but in vivo GRP75 overexpression reduced infarct size and protected against mitochondrial damage in a rat middle cerebral artery occlusion model of stroke (Xu et al., 2009) and a rat model of intracerebral hemorrhage (ICH) (Lv et al., 2017). GRP75 expression was also decreased in the mitochondrial fraction isolated from the SNpc of PD patients compared to controls (Jin et al., 2006). The above results suggest that GRP75 may be either beneficial or harmful in different pathogenic contexts, although the exact mechanisms are still unknown (Shi et al., 2008). One possible explanation is provided by reports that GRP75 interacted with MAM-associated α-syn in a DAergic cell line with strong membrane attachment affinity due to a high lipid:protein ratio (Zabrocki et al., 2008;Fantini and Yahi, 2011).
The MAM tethering proteins mitofusions Mfn1 and Mfn2 are dynamin-related GTPases responsible for membrane fusion via a large cytosolic GTPase domain embedded in the outer mitochondrial membrane (Rojo et al., 2002;Suarez-Rivero et al., 2016). Mfn2 heterodimerizes with Mfn1 to link ER and mitochondria to regulate organelle tethering (de Brito and Scorrano, 2008;Chen et al., 2012). Our study suggests that α-syn can affect mitochondrial morphology and modulate mitochondrial dynamics through reduction of Mfn1-and Mfn2dependent tethering in SH-SY5Y cells and mouse brain (Xie and Chung, 2012). Moreover, these findings identify MAM interface and inter-organelle contact disruption as novel mechanisms of ROT-induced α-syn toxicity.
Many studies rely on artificially overexpressed or recombinantly tagged proteins to investigate the underlying signaling mechanisms in PD. However, in the present study, we compared ROT-induced toxicity among cultures under different nutrient availability conditions and in vivo to further elucidate the potential mechanisms of α-syn neurotoxicity. As expected, ROT decreased TH and concomitantly increased α-syn accumulation, indicating degeneration of dopaminergic neurons both in vitro and in vivo. The nutrient-sensing mTOR and AMPK pathways appear to form a negative feedback loop that regulates the extent of ROT toxicity. More importantly, the present study implies that ER and MAM tethering proteins are also intimately involved in ROT-induced neurodegeneration. From these findings, we conclude that ROTtreated cell culture systems and mouse models are limited for recapitulating the clinical and pathological phenotypes of PD. We also conclude that serum concentration in culture medium can greatly influence the effects of ROT on cell growth, oxidative stress, and the various underlying signaling pathways. Further research in vitro and in vivo is necessary to establish stronger links between ROT-induced pathogenic mechanisms and human PD.

DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the manuscript/Supplementary Files.

ETHICS STATEMENT
All animal experiments were approved by the Ethical Committee of Animal Research of the DGIST, Daegu, South Korea accordance with international guidelines.

AUTHOR CONTRIBUTIONS
MR and Y-IL conceived and designed the study, and wrote the manuscript. MR performed the major experiments. Y-JH helped to perform the animal experiments. MR analyzed the data. All authors read and approved the manuscript for publication.


s2fieldsofstudy: Biology Medicine

text:
Growth Hormone-Releasing Hormone Receptor Antagonist Modulates Lung Inflammation and Fibrosis due to Bleomycin

Purpose Growth hormone-releasing hormone (GHRH) is a 44-amino acid peptide that regulates growth hormone (GH) secretion. We hypothesized that a GHRH receptor (GHRH-R) antagonist, MIA-602, would inhibit bleomycin-induced lung inflammation and/or fibrosis in C57Bl/6J mice. Methods We tested whether MIA-602 (5 μg or vehicle given subcutaneously [SC] on days 1–21) would decrease lung inflammation (at day 14) and/or fibrosis (at day 28) in mice treated with intraperitoneal (IP) bleomycin (0.8 units on days 1, 3, 7, 10, 14, and 21). Bleomycin resulted in inflammation and fibrosis around airways and vessels evident histologically at days 14 and 28. Results Inflammation (histopathologic scores assessed blindly) was visibly less evident in mice treated with MIA-602 for 14 days. After 28 days, lung hydroxyproline (HP) content increased significantly in mice treated with vehicle; in contrast, lung HP did not increase significantly compared to naïve controls in mice treated with GHRH-R antagonist. GHRH-R antagonist increased basal and maximal oxygen consumption of cultured lung fibroblasts. Multiple genes related to chemotaxis, IL-1, chemokines, regulation of inflammation, and extracellular signal–regulated kinases (ERK) were upregulated in lungs of mice treated with bleomycin and MIA-602. MIA-602 also prominently suppressed multiple genes related to the cellular immune response including those for T-cell differentiation, receptor signaling, activation, and cytokine production. Conclusions MIA-602 reduced lung inflammation and fibrosis due to bleomycin. Multiple genes related to immune response and T-cell functions were downregulated, supporting the view that MIA-602 can modulate the cellular immune response to bleomycin lung injury.

Introduction
Idiopathic pulmonary fibrosis (IPF) is the paradigm of fibrosing interstitial lung diseases. It occurs more commonly in aging males, and often has a limited survival time of 3-5 years (median 3.8 years) after diagnosis [1]. Although the disease is of unknown etiology, it is clearly related to specific genetic abnormalities (e.g., MUC5B, SFTPC, and others) and environmental factors (e.g., dust and smoking) [2]. In response to injury, fibroblasts proliferate and migrate into the lung. They synthesize extracellular matrix, providing a platform for further cellular growth [3]. Myofibroblasts secrete cytokines, such as TGF-ß, with autocrine and paracrine effects that drive fibrosis in the lung [4].
Growth hormone-releasing hormone (GHRH) is secreted primarily from the hypothalamus, but various other tissues can produce it locally [5]. GHRH stimulates the secretion and release of growth hormone (GH) by the pituitary and in turn regulates the secretion of GH and insulin-like growth factor 1 (IGF-1) through the pituitary GH/hepatic IGF-1 axis [6]. We have found expression of pituitary-type GHRH receptor (pGHRH-R) in normal human and IPF lung tissue by western blotting, suggesting that GHRH or GH could participate in lung development, growth, and repair [7].
Human fibroblasts express GHRH receptors, which stimulate proliferation of fibroblasts through GH/IGF-1-mediated signaling. When skin wounds in mice are exposed to GHRH agonist, fibroblasts increase and repair of epithelium is accelerated [12]. GHRH stimulates the expression of α-smooth muscle actin (αSMA), which confers contractile activity in myofibroblasts [13]. In addition to its effects on GH and IGF-1, GHRH-R antagonist MIA-602 inhibits signaling pathways, including PAK1-STAT3/NF-κB in gastric cancer cells, suggesting it could modulate inflammatory and fibrotic processes [14].
We therefore used an established bleomycin model of lung inflammation and fibrosis in C57/Bl6 mice and synthetic GHRH receptor antagonist MIA-602 to test whether inhibition of GHRH receptors would limit inflammation and/ or fibrosis.

Experimental Animals
We used 8-week-old C57Bl/6J male mice (The Jackson Laboratory, Bar Harbor, ME) in these experiments. Mice weighed about 26 g at the start. This protocol was approved by the Miami VAHS Animal Studies Subcommittee (IACUC) and it conformed to PHS Policy. Mice were randomly allocated to the experimental groups and housed in identical filter top cages in a ventilated rack. Mice were exposed to a 12-h light/dark cycle and had free access to standard laboratory chow and water.

Tissue Preparations
Mice were killed by CO 2 inhalation before any treatment, or on days 14 or 28 for micro-CT scans of lungs and harvesting of lung tissue [16]. The right mainstem bronchus was ligated and the right lung removed and frozen at -80 °C for hydroxyproline (HP) assays. The left lung was filled with 10% buffered formalin at ~ 25 cm H 2 O pressure, the bronchus ligated, and the lung fixed in formalin. Fixed lungs were embedded in wax and 5 μm sections stained with hematoxylin and eosin (H&E) or Masson's trichrome stain.

Lung Histopathology
Fibrosis was quantified in trichrome-stained sections using a modification of the Ashcroft score that describes grades of fibrosis [17]. Ten randomly chosen fields on each slide were scored as described and averaged. Inflammation in lung tissue was quantified in H&E-stained sections.

Hydroxyproline Assay
Right lungs were weighed and homogenized in 10 volumes of distilled water. Homogenates were hydrolyzed in 12 M HCl at 120 °C for 3 h as previously described [18]. Hydroxyproline was measured colorimetrically (560 nm) after hydrolysis using an assay kit (MAK008, Sigma-Aldrich, St. Louis, MO).

Micro-CT
Mice in each group were assessed by micro-CT scans (Bruker SkyScan 1176 Low-Dose Micro-CT, Knotich, Belgium) of the lungs after CO 2 inhalation. A tracheostomy tube was inserted and lungs inflated with air. Scans were examined qualitatively to confirm development of infiltrates and reticular densities.

Lung Fibroblasts
Newborn mouse lung fibroblasts (Mlg 2908) were obtained from the American Type Culture Collection, Manassas, VA and cultured in Eagle's minimal essential medium. Cells were grown in 6-well plates before incubation with 1 or 5 µM MIA-602 or vehicle, RNA isolation, and oxygen consumption measurements.

Annexin V-Propidium Iodide Assay
Apoptosis and necrosis were assessed with annexin V/propidium iodide staining (Annexin V: FITC Assay Kit, Bio Rad, Hercules, CA 94547) of lung fibroblasts incubated in 0, 1, or 5 µM MIA-602 for 24 h [19]. Culture medium and trypsinized cells were collected and centrifuged at 400×g for 5 min. The pellet was resuspended in 100 µL annexin V/propidium iodide. The suspension was incubated at 37 °C for 20 min, then washed with PBS, and resuspended in 500 µL PBS and fluorescence quantified by Beckman Coulter Life Sciences CytoFLEX benchtop flow cytometer (Beckman Coulter, Inc., Brea, CA).

RNA Isolation
RNA was extracted from fixed lung tissue in paraffin blocks using a Quick-RNA™ FFPE Kit (R1008, Zymo Research, Irvine, CA) following the manufacturer's protocol [20]. Samples were deparaffinized, digested with proteinase K, and decrosslinked at 65 °C for 15 min. RNA lysis buffer was added and mixed with ethanol. The mixtures were transferred to spin columns to isolate total RNA. RNA was extracted from cultured fibroblasts using a Direct-zol RNA MicroPrep kit (R2060; Zymo Research, Irvine, CA). Cells were washed with PBS and lysed in TRI reagent™, then purified using Direct-zol RNA columns. DNase I treatment was done in columns and RNA eluted in DNase/Rnase-free water.

Cellular Respiration
We measured effects of MIA-602 on mouse lung fibroblast oxygen consumption using the Agilent Seahorse XF Cell Mito Stress Test (Agilent Technologies, Santa Clara, CA) [21]. Fibroblasts were incubated with vehicle, 1 or 5 µM MIA-602 for 24 h before measurement of oxygen consumption. One day before assay 80,000 fibroblasts were seeded into Seahorse 24-well plates (n = 6 wells per condition). Basal respiration was established and oligomycin, FCCP, and rotenone plus antimycin A were added sequentially to measure ATP production, uncoupled respiration, and non-mitochondrial oxygen consumption [22].

RNA-seq and Pathway Analyses
At least 10 ng of total RNA was used as input for the KAPA RNA HyperPrep Kit with RiboErase (HMR) to create ribosomal RNA-depleted sequencing libraries, including sample indexing, to allow for multiplexing. Cluster generation and sequencing was done on the Illumina cBOT and HiSeq 3000 using reagents provided by Illumina, finally generating > 32 million single-end 100 base reads per sample.
We created de-multiplexed FASTQ files with Illumina supplied scripts in BCL2FASTQ software (v2.17). Illumina adapters were trimmed using the Trim Galore! package and aligned to the mouse reference genome (mm10) with STAR aligner (v2.5.0a) with default alignment parameters [23].
Gene counts were normalized using trimmed mean of M-values (TMM) method. Differential expression between groups was calculated with the exact test implemented in edgeR [24].
Pathway enrichment analyses were completed using Enrichr online and DAVID bioinformatics resource [25,26].

Data Analysis
Data are reported as arithmetic means ± SEM or SD as indicated. Medians and 25-75th percentiles were used to describe the distribution of histopathological scores. ANOVA followed by Dunnett's test or the Bonferroni correction was used for multiple comparisons with a control or among groups [27]. We planned at least six mice available at each time point in each group, so that we would have sufficient power to detect a 20% change in lung hydroxyproline given an assumed coefficient of variance of 0.2. p ≤ 0.05 was considered significant.

Micro-CT Scans
Mice treated with vehicle developed patchy infiltrative densities that persisted to day 28. Mice that received bleomycin plus MIA-602, the GHRH-R antagonist, appeared to have less prominent infiltrative densities in their lungs.

Lung Hydroxyproline Contents
Lung HP content did not increase significantly after 14 days of intermittent treatment with bleomycin in mice receiving MIA-602 or vehicle. However, after 28 days, lung HP content increased significantly in bleomycin-treated mice that received vehicle, but not in bleomycin-treated mice that received MIA-602 on days 1-21. The data are summarized in Fig. 1.

Lung Histopathology
After 14 days of intermittent bleomycin, inflammation and patchy mild fibrosis were qualitatively evident in mouse lungs. More extensive fibrosis and fewer inflammatory cells were evident after 28 days. Both inflammatory changes and fibrosis appeared decreased in mice that received MIA-602 in addition to bleomycin. Representative examples are shown in Fig. 2.

Cellular Respiration
In vitro, MIA-602 at 5 µM concentration in complete medium increased fibroblast basal respiration and maximal respiration (after FCCP), compared to vehicle. Both 1 and 5 µM MIA-602 also appeared to increase non-mitochondrial respiration (after antimycin A and rotenone). These data are summarized in Fig. 3. with Masson's trichrome stain and assessed semi-quantitatively for inflammation and fibrosis as described in the text. 14 days after bleomycin was started, cellular inflammation and early fibrosis was detected in lungs of mice treated with bleomycin and vehicle. Less inflammation appeared to be present in lungs of mice that received bleomycin and MIA-602 (middle panels). 28 days after bleomycin was started, increased fibrosis was evident in lungs of mice treated with bleomycin and vehicle. Less fibrosis appeared to be present in lungs of mice that received bleomycin and MIA-602, the growth hormone receptor antagonist

RNA-seq Gene Expression
We explored the effects of MIA-602 or vehicle on gene expression in lungs from mice treated in vivo with bleomycin for 28 days (data not shown). Several physiologically relevant genes were expressed differently after treatment with bleomycin (absolute fold change > 1.5 and FDR < 0.01). Specifically, after 28 days during which bleomycin was administered, genes related to the extracellular matrix, Wnt regulation and signaling, and the extracellular region were upregulated, consistent with known effects of bleomycin. We found several relevant genes to be downregulated by bleomycin, including those related to lung morphogenesis and development, extracellular matrix organization, and alveolar septal development.
Transcriptome profiles then showed numerous genes expressed differently after treatment with MIA-602. Inversely modulated genes were highly enriched in pathways related to the adaptive immune response, T-cell differentiation, T-cell signaling, extracellular matrix organization, T-cell activation and differentiation, and cytokine production, all consistent with the putative anti-inflammatory and anti-fibrotic effects of MIA-602. The ten most differentially expressed genes detected in lung tissue treated with MIA-602 compared to vehicle treatment are shown in Table 1. Those genes differentially expressed in fibrotic lungs from bleomycin-exposed mice treated with MIA-602 compared to vehicle are displayed as a heat map in Fig. 4.
Similarly, we directly tested the in vitro effects of 1 and 5 µM MIA-602 on normal mouse lung fibroblasts (not Fig. 3 GHRH-R antagonist increases lung fibroblast basal and maximal oxygen consumption. Panel A shows a representative mitochondrial stress assay of mouse lung fibroblasts (data shown are means ± SD of 6 wells in each condition) exposed to vehicle (light gray), 1 µM (medium gray) or 5 µM (black) MIA-602 for 24 h before measurements of oxygen consumption with oligomycin, FCCP, antimycin A, and rotenone. 5 µM MIA-602 increased basal oxygen consumption (*p = 0.0403 compared to vehicle) and maximal uncoupled respiration (**p < 0.0001) of normal mouse lung fibroblasts. Panel B shows that 5 μM MIA-602 increased both basal respiration (*p = 0.0125) and spare respiratory capacity (**p < 0.0001)

Discussion
Pathophysiological GH secretion and IGF-1 activation have growth-promoting effects in the lung resulting in increased alveolar size [28]. IGF-1 itself increases α-smooth muscle actin in lung fibroblasts and promotes a myofibroblast phenotype. The pituitary-type GHRH receptor is present in both normal and IPF lung tissue [7], suggesting that local secretion of GH may occur physiologically and have direct effects on lung tissue. MIA-602 partially inhibits both lung inflammation and fibrosis, assessed histopathologically and biochemically, after intraperitoneal bleomycin. RNA-seq data, importantly, show suppression of the adaptive immune response, T-cell differentiation and activation, and cytokine production by MIA-602 in bleomycin-treated mouse lungs. The effects of the GHRH-R antagonist we observe have implications for fibrosing lung diseases in humans, and they could, importantly, reveal novel pathways amenable to clinical drug development [29].
We initially confirmed development of lung infiltrates due to bleomycin with micro-CT scans, inflammation, and fibrosis with histopathological examination, and increased collagen with biochemical assays. Similar models have been used successfully in the development of anti-fibrotic drugs, including pirfenidone and nintedanib.
Senescent fibroblasts that display respiratory abnormalities, indicating mitochondrial damage, express both STAT3 and p21 as markers of the senescent phenotype [30]. MIA-602 downregulates p21-activated kinase and STAT3 and NFκB in gastric cancer cells [14]. GHRH antagonists like MIA-602 could modulate the senescent phenotype leading to fibrosis, and conceivably be one of the mechanisms that lessens the fibrotic response in this model.
In vitro, MIA-602 at micromolar concentrations increased basal and maximal mitochondrial respiration, and it resulted in marked cytolytic death of mouse lung fibroblasts. Mitochondrial dysfunction and loss of apoptotic potential occur in fibroblasts from IPF lungs, and enhancement of mitochondrial function itself by MIA-602 at lower concentrations in vivo might modulate fibrosis by maintaining the capacity for mitophagy and apoptosis [31].
GHRH-R antagonists decrease lipid peroxidation, protein carbonyls, and nitrotyrosine in prostate cancer cells [32], indicating antioxidant effects that would augment their other anti-inflammatory effects [33]. Both GH and IGF-1 stimulate neutrophil superoxide (O 2 − ) production [9]. Since GHRH-R antagonists inhibit GH secretion and IGF-1 activation [34], it could be predicted that they would inhibit both O 2 − and hydrogen peroxide (H 2 O 2 ) release during the inflammatory phase of injury [35]. Cellular levels of oxidant stress are decreased by MIA-602, and its antioxidant effect would limit redox signaling in response to receptor ligation [11].
MIA-602 disrupts the PI3K/AKT pathway in several experimental systems. PI3K/AKT signaling is involved in the pathogenesis of bleomycin-induced fibrosis [36], and suppression of the PI3K/AKT pathway by GHRH antagonist could also lessen lung fibrosis. GHRH clearly appears involved in the lung's response to treatment with bleomycin and subsequent healing.
Genes related to the extracellular matrix and the Wnt pathway were over expressed in fibrotic lungs from mice treated with bleomycin, compared to gene expression in naïve controls [37]. In contrast, genes related to epithelial tube branching, lung morphogenesis, and lung development were under expressed after bleomycin and development of fibrosis. Genes related the immune response, cellular adhesion, and remodeling, and T-cell signaling were also found to be upregulated in lungs from rats treated with TGF-ß [38].
T cells play a central role in the lungs of patients with pulmonary fibrosis [39]. T-cell receptors and costimulatory molecules are required for activation of T cells and in development of inflammation-driven lung fibrosis [40]. We found that downregulation of T-cell receptor complex genes (CD3E, CD3G, CD4, and CD8A) had the highest associations in pathway analyses. MIA-602 may thus play an important role in lung tissue by modification of  T-cell signaling and potentially reducing inflammation and fibrosis. These data show that GHRH-R is present in human lungs; and, in a relevant in vivo model, lung fibrosis is modulated by its inhibition. Functional findings implicate GHRH and GH in fibrosing lung disease, and they are consistent with demonstrated effects of the GHRH-R antagonist on mitochondrial respiration and fibroblast cytotoxicity. MIA-602 inhibits intracellular signaling pathways, including p21-activated kinase/STAT3/NFκB and PI3K/AKT, in addition to having intrinsic antioxidant activity. Further, it could support mitochondrial function and maintain autophagy, minimizing fibrosis. These findings suggest the merit of further investigations of GHRH-R antagonists like MIA-602 regarding their mechanisms of action and applications in management of pulmonary fibrosis.