Full Length Article
Methotrexate negatively acts on inflammatory responses triggered in Drosophila larva with hyperactive JAK/STAT pathway

https://doi.org/10.1016/j.dci.2021.104161Get rights and content

Highlights

  • MTX treatment results in delayed pupariation, eclosion, reduced climbing ability and fusion of ommatidia in Drosophila.

  • MTX downregulates the expression of the JAK/STAT pathway components activated upon wasp infestation.

  • MTX inhibits the tumor penetrance and expressivity in hopTum-l mutants.

  • MTX reduces the gene expression of upd3hop, Stat92E, Tep2 and Tep4 in hopTum-l mutants.

  • MTX affects the lamellocytes formed in response to both the acute and the chronic inflammatory conditions.

Abstract

Drosophila is a valuable paradigm for studying tumorigenesis and cancer. Mutations causing hematopoietic aberrations and melanotic-blood-tumors found in Drosophila mutants are vastly studied. Clear understanding about the blood cells, signaling pathways and the tissues affected during hematopoietic tumor formation provide an opportunity to delineate the effects of cancer therapeutics. Using this simple hematopoietic archetype, we elucidated the effects of the anti-cancer drug, Methotrexate (MTX) on immune responses in two scenarios i.e. against wasp infection and in hematopoietic mutant, hopTum-l. Through this in vivo study we show that MTX impedes the immune responses against wasp infection including the encapsulation response. We further observed that MTX reduces the tumor penetrance in gain-of-function mutants of JAK/STAT pathway, hopTum-l. MTX is anti-inflammatory as it hinders not only the immune responses of acute inflammation as observed after wasp infestation, but also chronic inflammatory responses associated with constitutively activated JAK/STAT pathway mutant (hopTum-l) carrying blood tumors.

Introduction

Therapeutic agents targeting cancer causing molecules have successfully directed decades of promising research (Sawyers, 2004). Yet, existing cancer therapeutics have failed to satisfy the requirements for an effective treatment with no side effects (Maeda and Khatami, 2018). Chemotherapy is a systemic treatment designed to target exclusively the cancer cells that can either destroy, shrink and/or regulate these cells. But chemotherapy also poses a disadvantage as these drugs are ineffectual in completely distinguishing between cancerous and non-cancerous cells (Bagnyukova et al., 2010; Sutradhar and Amin, 2014). The expected effect of chemotherapy is on metastasizing cancer cells and it ensures that the treatment either controls, cures or is palliative. Understanding how the chemotherapeutic drugs affect the wild type cells can provide insights on the cellular mechanisms perturbed upon its treatment. Past few decades have contributed to the knowledge on drug repositioning and repurposing. In this regard using model organisms is highly beneficial to determine the in vivo effects of anti-cancer drugs.

While laboratory mouse is most frequently used to evaluate anti-cancer drugs, past few decades of cancer research using other model organisms such as Drosophila and Danio rerio proved to be highly advantageous (Gao et al., 2014; Hogenesch and Nikitin, 2012). Drosophila shares similarities with mammals in the development of blood cells that are evolutionary conserved (Gautam et al., 2021). Drosophila larval hemocytes are found in either circulating hemolymph (originating from head mesoderm) or the hematopoietic organ the lymph gland (originating in the cardiac mesoderm of the embryo) (Banerjee et al., 2019). To date three matured blood cells are detected in the hemolymph and the lymph gland. Blood cells in circulation are either sessile or freely floating in hemolymph. These matured blood cells include plasmatocytes (>95%), crystal cells (>5%) and the lamellocytes (<1%). While acquired immunity does not prevail in Drosophila, the conventional innate immune responses including some of the signaling pathways and the mechanisms involved are conserved between fruit flies and mammals (Crozatier and Meister, 2007; Lemaitre and Hoffmann, 2007). Blood cells are immune defensive and in Drosophila they orchestrate the anti-inflammatory effects by host defense mechanisms such as phagocytosis and synthesis of anti-microbial peptides (Vlisidou and Wood, 2015). In response to wasp infection, in Drosophila larva a multifactorial network of signaling cascades is turned on, leading to the constitutive division of hemocytes. Upon wasp infection the host identifies the parasite as foreign body leading to the encapsulation of the parasite. Some of the precursor cells found in both, the circulating hemolymph and the anterior lobes of hematopoietic organ differentiate into lamellocytes. Parasitoid egg is encapsulated by the blood cells necessitating the involvement of the large (>20 μm) lamellocytes (Schlenke et al., 2007; Small et al., 2012). The encapsulated bodies are thereby out-casted within the hemolymph. Increased number of lamellocytes in circulation is observed as a consequence of genetic mutations as well that perturb hematopoietic signaling such as JAK/STAT, Toll/NF-κB, RAS etc. (Babcock et al., 2008; Banerjee et al., 2019; Zettervall et al., 2004). Loss of function (LOF) mutations of the negative regulators or the gain of function (GOF) mutations in positive regulators of the hematopoietic pathways escalate the rate of blood cell division and melanotic microtumor formation. LOF mutation of Ubc9 and Cactus results in the upregulation of the Toll/NF-κB pathway while in the GOF mutants hopTum-l JAK/STAT pathway is constitutively upregulated. Melanotic tumors found in the circulating hemolymph of these hematopoietic mutants, are cellular outgrowths formed from the larval hemocytes with some degree of melanization (Rizki et al., 1957; Rizki and Rizki, 1983, 1984). In Drosophila larva JAK-STAT pathway is primarily responsible for the differentiation of lamellocytes (Vlisidou and Wood, 2015). Tumorous lethal (Tum-l) is an oncogene and larvae carrying this mutation display defective hematopoiesis. hopTum-l mutants carrying a dominant mutation in the locus of the hopscotch gene manifest hematopoietic neoplasm due to the uncontrolled blood cell division, differentiation and expanded population of lamellocytes (Hanratty and Dearolf, 1993). Upon injecting the over-proliferating and highly differentiated hopTum-l lymph glands into the wild type Drosophila adults (females) the injected tissue was capable of inducing melanization and growth of the blood cells. These blood cells shared identities with the parent hematopoietic tissue hopTum-l establishing neoplastic nature of these melanotic tumors (Hanratty and Ryerse, 1981).

Various studies depicted the strategies employed by the anti-inflammatory and anti-cancer drugs to target JAK/STAT pathway components for their role in inflammation and tumorigenesis. Aspirin reduced the over-proliferation of blood cells in hopTum-l (GOF) mutants and improved their viability (Panettieri et al., 2019). Experimental evidences (in vitro) demonstrated that the known anti-folate drug MTX behaved like an anti-JAK/STAT due to its inhibitory effect on the phosphorylation of Stat92E, the transcription factor of the JAK/STAT pathway (Thomas et al., 2015). They further showed that the effect of MTX on JAK/STAT pathway still persisted even in the presence of folinic acid iterating that MTX's potential to reduce the phosphorylation of Stat5 is not completely dependent on folinic acid. These results implicate unidentified mechanism through which MTX exerts its effects in the target cells (Thomas et al., 2015). MTX is one of the Food and Drug Administration (FDA) approved anti-metabolite chemotherapeutic drug (Hannoodee and Mittal, 2021). MTX inhibits Dihydrofolate reductase (DHFR) via competitive inhibition with 1000-fold more affinity compared to folate. DHFR is functionally involved in the conversion of Dihydrofolate to active Tetrahydrofolate (reduced folate factors) (Rajagopalan et al., 2002). HD-MTX (high dose MTX) is used as a chemotherapeutic and LD-MTX (low dose MTX) is shown to be an anti-inflammatory prescribed for immunoinflammatory rheumatological diseases (Malaviya et al., 2010). One of the side effects of low dose MTX (LD-MTX) is cytopenia (Gutierrez-Ureña et al., 1996). Importantly, upon treatment with MTX there is a reduction in the white blood cell (WBC) numbers (Sosin and Handa, 2003). WBCs are required for eliciting the immunoinflammatory responses during infection (Cioffi et al., 1993). Fruit flies fight off infections (bacterial, viral or other foreign pathogens) using plasmatocytes (macrophages) (Gold and Brückner, 2015). While lamellocytes are specialized in encapsulating large foreign bodies that cannot be phagocytized (Rizki and Rizki, 1992). Many key questions related to the drug (anti-cancer or anti-inflammatory) effects on hematopoietic pathways triggered in the blood cells against bacterial or parasitoid infections are yet to be completely understood. Therefore, Drosophila is an ideal model to decipher the effects of MTX on blood cells with immune functions involved in fighting infections. We can further benefit from studying the hematopoietic signaling pathways affected in these blood cells upon treatment with MTX. As Drosophila larva exhibit a simple hematopoietic system with only three mature blood cells (Wang et al., 2013) understanding the morphological and genetic changes in these blood cells is faster and easier unlike the mammalian model with a complex network of signaling pathways regulating hematopoiesis.

Through this study we bring to focus the significance of MTX on both, the blood cells’ mediated inflammatory responses through JAK/STAT pathway and blood tumors observed in JAK/STAT pathway mutants, hopTum-l. We studied the role of MTX on the immune cells and the inflammatory responses associated with blood tumors. Due to the effects of MTX on JAK/STAT pathway we hypothesized that formation of blood tumors with excessive lamellocytes observed in hopTum-l mutants can be intervened by the treatment with the anti-cancer drug, MTX. We therefore elucidate MTX effects in fly hosts with upregulated JAK/STAT pathway causing abnormal hematopoiesis in two different conditions, first scenario is after wasp infestation and the second observed in hematopoietic mutants, hopTum-l. Parasitization by the Leptopilina boulardi (strain Lb17) triggers acute inflammatory responses in the host body to combat infection (Hetru and Hoffmann, 2009; Paddibhatla et al., 2010). These include the formation of lamellocytes, activation of Toll/NF-κB signaling pathways in the immune cells and synthesis of AMPs such as Drosomycin. These immune responses are categorized under acute inflammation regulated through Toll pathway. After encapsulation of parasitoid wasp egg which is the final step to combat parasitoid infestation these responses are downregulated. While in constitutively activated hematopoietic signaling as observed in mutants of hopTum-l and Ubc9−/− the fly larva encounters an autofeedback loop established in the immune cells (fat body and blood cells) due to aberrant signaling synonymous to chronic inflammation (Paddibhatla et al., 2010). Through our study we address for the first time the effects of MTX on these inflammatory responses caused due to hematopoietic deregulation observed after parasitization (acute) and in hematopoietic mutants, hopTum-l (chronic).

Section snippets

Materials and methods

Drosophila stocks: All the stocks of Drosophila were raised on the standard protocol for cornmeal-malt-agar-yeast media at 25 °C under 12h:12h light-dark cycles. We used wild type Drosophila melanogaster (Canton S) and hopTum-l, gain of function mutants of JAK/STAT pathway, [Gift from Dr. Shubha Govind's laboratory, City College, CUNY (Panettieri et al., 2019; Piper et al., 2014).

Wasp stock and infection: Leptopilina boulardi (strain Lb17). Standard protocol was used for rearing wasps on the

Methotrexate treatment delays development and affects locomotion in Drosophila

To comprehend if the larvae fed with MTX are consuming the fly food we first performed “food coloring assay”. We prepared the fly food with both coloring agent and MTX as mentioned in the material and methods (Coloring agent assay). We used 250 mg of coloring agent and mixed it into a MTX solution of 6.67 μM as starting higher concentration and further concentrations were determined similar to the procedure used to make MTX dilutions. Drug was administered along with the fly food (Figure figs1

Methotrexate impedes chronic inflammation in hopTum-l

Since MTX had an effect on the acute arm of inflammation in Drosophila we also wanted to verify the effect of MTX on the other arm of inflammation i.e., chronic inflammation. To decipher this, we utilized hopTum-l mutants that manifest an uncontrolled over proliferation of blood cells due to constitutively active JAK/STAT pathway. This condition is synonymous to chronic inflammation with the third instar larva having defective blood cell regulation, fat body tissue and melanotic masses observed

Discussion

Substantial number of studies have explicitly demonstrated the side effects of chemotherapeutics targeting non-cancerous cells. It is imperative to unravel the exact mechanisms involved in chemotherapy that not only inhibit the cancer growth and but also avert side effects. Methotrexate is used for the treatment of leukemia and several other auto-immune diseases such as rheumatoid arthritis (RA), psoriasis, crohn's disease etc. Innumerable studies existing using mammalian models that

Conclusions

Various fields of medicine, pharmacology, biotechnology and therapeutic fields of clinical research are contributing to the immense research addressing various diseases including cancer. Drug discovery is a cumbersome, lengthy and high-investment procedure with various challenges that are difficult to overcome such as lack of knowledge about the pathophysiology of disease under investigation and poorer understanding of heterogeneity of the patients under observation. Past decade shed light on

Declaration of competing interest

Authors share no competing or financial interest.

Acknowledgements

We are grateful to Prof. Shubha Govind for providing fly stocks and Lb17 wasps strains. We thank Prof. Istavan Ando for sharing the L1/Atilla antibody. We acknowledge Ms. Anuradha Venkatakrishnan Chimata for her valuable feedback and critical insights on the manuscript. We are grateful to Bloomington Drosophila Stock Center for fly strains. We thank the central confocal facility at the University of Hyderabad (Plant Sciences Department and the Nanotechnology Department).

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