The activity of cathepsin B was examined using the Fluorometric ab65300 assay kit. Fasciola gigantica-derived rFgCatB enzymatic activity was determined by measuring its ability to cleave the fluorescent synthetic substrate RR-AFC to release free AFC. The results showed that the enzyme activity of rFgCatB is several fold higher than that of the control, confirming the functional activity for cathepsin B (**Figure 2**).

### Binding Affinity of rFgCatB Protein to Goat PBMCs

Indirect immunofluorescence staining was used to determine the binding affinity of rFgCatB protein to the surface of PBMCs. By incubating rFgCatB-treated PBMCs with rabbit anti-rFgCatB antibodies, the localization of the red Cy3 conjugated goat antirabbit IgG secondary antibody on the cell surface was observed, suggesting successful binding of rFgCatB to the surface of PBMCs (**Figure 3**). There was no fluorescence observed in the untreated control cells.

### rFgCatB Protein Increased Cytokine Production

To understand how rFgCatB modulates cytokine production of PBMCs, the levels of six cytokines, IL-2, IL-4, IL-10, IL-17, IFN-γ, and TGF-β, were determined. As shown in **Figure 4**, when PBMCs were treated with serial concentrations of rFgCatB protein, the production of all six cytokines was significantly increased compared with control (PBS-treated) PBMCs.

### Cytotoxic Effect of rFgCatB Protein

We examined whether rFgCatB protein affects the viability of PBMCs. The CCK-8 assay showed that the viability of PBMCs

was remarkably decreased following treatment with rFgCatB protein, at all tested protein concentrations (**Figure 5**).

## Nitric Oxide (NO) Production

As shown in **Figure 6**, compared to the control (PBS-treated PBMCs), NO release was slightly increased in rFgCatB-treated PBMCs at 40µg/ml and was significantly increased in rFgCatBtreated PBMCs at 80µg/ml, but not at 10 or 20 µg/ml.

### rFgCatB Protein Induced Cell Apoptosis

To explore whether rFgCatB protein induces apoptosis in goat PBMCs, Annexin V-FITC apoptosis assay was used. The rFgCatB protein significantly induced apoptosis in PBMCs at all tested concentrations compared to PBS-treated, control PBMCs (**Figure 7**). The apoptosis was induced in a dosedependent manner with the percentage of apoptotic cells treated with rFgCatB at 10, 20, 40, and 80µg/ml were 28.88 ± 2.631%, 30.95 ± 3.128%, 33.50 ± 2.152%, and 46.17 ± 5.955%, respectively.

### DISCUSSION

In this study, we cloned and expressed the gene encoding cathepsin B of F. gigantica in the methylotrophic yeast P. pastoris. Although the expected size of the purified rFgCatB protein with six-histidine tag is 38.2 kDa, a significant increase in its molecular weight was observed. Yeast expression system has been known to introduce post-translational modifications such as glycosylation which may affect protein folding. Also, recombinant proteins produced in P. pastoris tend to be

hypermannosylated. On SDS-PAGE gels, rFgCatB appeared as a group of bands with molecular weights between ∼36 and 70 kDa. The observed heterogeneity in the size of the protein may be attributed to N-linked glycosylation (19). Deglycosylation of rFgCatB using Endo H revealed ∼38 kDa band, which corresponds to the theoretical molecular mass of rFgCatB, and another band with molecular masse of 36 kDa, suggesting the presence of two different glycosylated species (i.e., diglycosylated forms) of rFgCatB, particularly, as both reacted in Western blot. This type of finding has been also reported for cathepsin B from the Asiatic liver fluke Opisthorchis viverrini (20).

Our results showed that rFgCatB induced expression of Th1 type cytokines (IL-2 and IFN-γ), Th2 type cytokines (IL-4, IL-10, and TGF-β), and Th17 type cytokine (IL-17), suggesting that rFgCatB can induce a mixed T helper 1 (Th1)-, Th2-, and Th17-type immune response. The high levels of pro-inflammatory cytokines (IL-2 and IFN-γ) and activation of monocytes have been associated with intestinal pathology and release of NO to limit the fluke growth (21–23). On the other hand, high expression of Th2 anti-inflammatory cytokines can facilitate parasite persistence, while minimizing host tissue damage (24–27). For example, the anti-inflammatory cytokine IL-4 inhibits

cytokines in a dose-dependent manner. Graphs represent means ± standard deviations of data from three independent biological replicates (IL-2 (10 µg/ml: F(5, 36) = 46.98, P < 0.0001; 20 µg/ml: F(5, 36) = 46.98, P < 0.0001; 40 µg/ml: F(5, 36) = 46.98, P < 0.0001; 80 µg/ml: F(5, 36) = 46.98, P < 0.0001; 160 µg/ml: F(5, 36) = 46.98, P < 0.0001), IL-4 (10 µg/ml: F(5, 36) = 43.65, P < 0.0001; 20 µg/ml: F(5, 36) = 43.65, P < 0.0001; 40 µg/ml: F(5, 36) = 43.65, P < 0.0001; 80 µg/ml: F(5, 36) = 43.65, P < 0.0001; 160 µg/ml: F(5, 36) = 43.65, P < 0.0001), IL-10 (10 µg/ml: F(5, 36) = 40.51, P = 0.0012; 20 µg/ml: F(5, 36) = 40.51, P < 0.0001; 40 µg/ml: F(5, 36) = 40.51, P < 0.0001; 80 µg/ml: F(5, 36) = 40.51, P < 0.0001; 160 µg/ml: F(5, 36) = 40.51, P < 0.0001), IL-17 (10 µg/ml: F(5, 36) = 77.39, P = 0.0002; 20 µg/ml: F(5, 36) = 77.39, P < 0.0001; 40 µg/ml: F(5, 36) = 77.39, P < 0.0001; 80 µg/ml: F(5, 36) = 77.39, P < 0.0001; 160 µg/ml: F(5, 36) = 77.39, P < 0.0001), TGF-β (10 µg/ml: F(5, 36) = 137.5, P < 0.0001; 20 µg/ml: F(5, 36) = 137.5, P < 0.0001; 40 µg/ml: F(5, 36) = 137.5, P < 0.0001; 80 µg/ml: F(5, 36) = 137.5, P < 0.0001; 160 µg/ml: F(5, 36) = 137.5, P < 0.0001), and IFN-γ (10 µg/ml: F(5, 36) = 96.82, P = 0.0003; 20 µg/ml: F(5, 36) = 96.82, P < 0.0001; 40 µg/ml: F(5, 36) = 96.82, P < 0.0001; 80 µg/ml: F(5, 36) = 96.82, P < 0.0001; 160 µg/ml: F(5, 36) = 96.82, P < 0.0001). Asterisks indicate statistical significance between treated and untreated control PBMCs (\*\*P < 0.01; \*\*\*P < 0.001; \*\*\*\*P < 0.0001 compared with control).

NO production (28) and promotes Th2 differentiation (29), thereby facilitating the production of other antiinflammatory cytokines (e.g., IL-10 and TGF-β) and inhibiting pro-inflammatory cytokines (e.g., IL-2 and IFNγ) (30, 31). Also, IL-10 decreases the production of IFN-γ and IL-2 (22, 31).

TGF-β, together with other inflammatory cytokines, can promote Th17 differentiation (32–34). Th17 cells play an important role in host protection against various parasitic infections by recruiting macrophages and neutrophils to infected tissues, and through the modulation of Th1/Th2 balance (34–36). The role of IL-17 in the inflammatory process during F. gigantica infection has been reported (37, 38). Interestingly, TGF-β can inhibit T cell proliferation by suppressing the production of IL-2, and inhibiting the differentiation of Th1 and Th2 cells (39).

We have previously shown that F. gigantica proteins, rFg14-3- 34 and rFgRab10, inhibit cell proliferation, and induce apoptosis and NO production in goat PBMCs (6, 7). The results of the present study lend further support to these previous findings, where rFgCatB was found to bind to the surface of PBMCs similar to what we have demonstrated for rFg14-3- 4 and rFgRab10 proteins (6, 7), and to reduce the viability and increase apoptosis of PBMCs. The biological relevance of the pro-apoptotic effect of rFgCatB on PBMCs remains to be determined. However, induction of apoptosis, rather than necrosis, may favor the parasite's persistence because apoptotic cell death does not provoke inflammatory response (40), which can be detrimental to the parasite's survival inside the host.

The antiproliferative and pro-apoptotic effects of E/S products of Fasciola spp. on immune cells are some of the strategies used by these liver flukes to hamper immune defenses, leaving the host more vulnerable to infection. F. hepatica-derived E/S products have been shown to inhibit the proliferation of sheep lymphoid cells, especially CD4+ T lymphocytes (41–43), reduce the proliferation of rat spleen mononuclear cells (44) and induce apoptosis of murine

FIGURE 6 | Effects of rFgCatB protein on the production of NO. PBMCs were treated with PBS or with serial concentrations of rFgCatB protein and maintained at 37◦C for 24 h. NO concentration was measured by Griess assay. Graphs represent means ± standard deviations of data from three independent biological replicates (10 µg/ml: ANOVA, F(4, 10) = 27.91, P = 0.9998; 20 µg/ml: ANOVA, F(4, 10) = 27.91, P = 0.9702; 40 µg/ml: ANOVA, F(4, 10) = 27.91, P = 0.0220; 80 µg/ml: ANOVA, F(4, 10) = 27.91, P < 0.0001). Asterisks indicate significant differences between rFgCatB-treated and PBS-treated control cells (\*p < 0.05; \*\*\*\*P < 0.0001; ns, non-significant compared with control).

FIGURE 7 | rFgCatB protein induced apoptosis in goat PBMCs. Annexin V/PI staining was used to quantify apoptotic cells by flow cytometry. (A) The FACS plot showing apoptosis of PBMCs in response to exposure to rFgCatB protein. (B) Apoptotic cells (Annexin V+/PI-) were plotted and compared with the percentage of cell population. Graphs represent means ± standard deviations of data from three independent biological replicates (10 µg/ml: ANOVA, F(4, 10) = 34.92, P = 0.0006; 20 µg/ml: ANOVA, F(4, 10) = 34.92, P = 0.0003; 40 µg/ml: ANOVA, F(4, 10) = 34.92, P < 0.0001; 80 µg/ml: ANOVA, F(4, 10) = 34.92, P < 0.0001). The asterisks indicate significant differences between rFgCatB-treated and PBS-treated control goat PBMCs (\*\*\*P < 0.001; \*\*\*\*P < 0.0001 compared with control).

eosinophils and peritoneal macrophages (45, 46). Also, immunosuppression of CD4+ T lymphocytes has been observed in F. hepatica-infected goats (47). Additionally, F. hepatica can induce apoptosis in sheep PBMCs by up-regulating the expression of TNF-α and TNFR1/TNFR2 (48). The induction of apoptosis in sheep eosinophils (49) and peritoneal leucocytes (50) has been suggested to play a role in the pathogenesis of F. hepatica by supporting the survival of the juvenile parasites during the migratory and biliary stages of infection.

In summary, our data show that rFgCatB interacts with serum from goats infected with F. gigantica and accumulates at the surface of PBMCs. The importance of our data resides in the fact that rFgCatB represents a new mechanism for F. gigantica to evade the host's immune response through modulation of the immune response and biological functions of PBMCs. Exposure of these cells to rFgCatB caused increased production of cytokines (IL-2, IL-4, IL-10, IL-17, TGF-β, and IFN-γ), increased NO production, increased apoptosis, and inhibition of cell viability. Our data provide a proof of concept that rFgCatB is involved F. gigantica-interaction with immune cells. In the light of these findings and given that rFgCatB and other F. giganticaderived proteins (e.g., rFg14-3-34 and rFgRab10) can modulate key cellular and immunological functions of goat PBMCs, future work should focus on identifying the appropriate synergistic combinations of these proteins to develop a cocktail vaccine for testing against F. gigantica infection.

### DATA AVAILABILITY

All datasets generated for this study are included in the manuscript.

### REFERENCES

### ETHICS STATEMENT

All experimental protocols were reviewed and approved by the Animal Administration and Ethics Committee of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Permit No. 2018-012). All animal experiments were performed in strict compliance with the Animal Ethics Procedures and Guidelines of the People's Republic of China. All efforts were made to minimize the suffering of animals, and daily health checks were performed during the entire experiments.

### AUTHOR CONTRIBUTIONS

X-QZ, XL, and HE conceived the idea, planned the experiments, and provided critical feedback. DC performed the experiments, analyzed the data, and drafted the manuscript with the help of HE. A-LT, J-LH, J-XL, XT, and X-DY participated in the implementation of the study. All authors read and approved the final manuscript.

### FUNDING

This work was supported by the National Key Basic Research Program (973 Program) of China (Grant No. 2015CB150300) and the Agricultural Science and Technology Innovation Program (ASTIP) (Grant No. CAAS-ASTIP-2016-LVRI-03).

### ACKNOWLEDGMENTS

We thank Professor Kevin Gough (University of Nottingham) and Professor David Buttle (University of Sheffield) for helpful comments and constructive suggestions.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Chen, Tian, Hou, Li, Tian, Yuan, Li, Elsheikha and Zhu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# IL-4 Mediated Resistance of BALB/c Mice to Visceral Leishmaniasis Is Independent of IL-4Rα Signaling via T Cells

Emma McFarlane1 , Thabang Mokgethi 1 , Paul M. Kaye2 , Ramona Hurdayal 3,4,5,6 \*, Frank Brombacher 3,4,5, James Alexander 1 and Katharine C. Carter 1 \*

*1 Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, United Kingdom, 2 Department of Biology, Centre for Immunology and Infection, Hull York Medical School, University of York, York, United Kingdom, 3 International Centre for Genetic Engineering and Biotechnology, Cape Town Component, Cape Town, South Africa, 4 Division of Immunology, Department of Pathology, Faculty of Health Sciences, Institute of Infectious Diseases and Molecular Medicine (IDM), South African Medical Research Council (SAMRC) on Immunology of Infectious Diseases, University of Cape Town, Cape Town, South Africa, 5 Faculty of Health Sciences, Wellcome Centre for Infectious Diseases Research in Africa, Institute of Infectious Diseases and Molecular Medicine (IDM), University of Cape Town, Cape Town, South Africa, 6 Department of Molecular and Cell Biology, University of Cape Town, Cape Town, South Africa*

#### *Edited by:*

*Jude Ezeh Uzonna, University of Manitoba, Canada*

#### *Reviewed by:*

*Gaurav Gupta, NIIT University, India Peter Epeh Kima, University of Florida, United States Abhay Satoskar, The Ohio State University, United States*

#### *\*Correspondence:*

*Ramona Hurdayal<EMAIL_ADDRESS>Katharine C. Carter<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Microbial Immunology, a section of the journal Frontiers in Immunology*

*Received: 14 June 2019 Accepted: 02 August 2019 Published: 16 August 2019*

### *Citation:*

*McFarlane E, Mokgethi T, Kaye PM, Hurdayal R, Brombacher F, Alexander J and Carter KC (2019) IL-4 Mediated Resistance of BALB/c Mice to Visceral Leishmaniasis Is Independent of IL-4R*α *Signaling via T Cells. Front. Immunol. 10:1957. doi: 10.3389/fimmu.2019.01957* Previous studies infecting global IL-4Rα −/−, IL-4−/−, and IL-13−/−mice on a BALB/c background with the visceralizing parasite *Leishmania donovani* have shown that the T helper 2 cytokines, IL-4, and IL-13, play influential but not completely overlapping roles in controlling primary infection. Subsequently, using macrophage/neutrophil-specific IL-4Rα deficient BALB/c mice, we demonstrated that macrophage/neutrophil unresponsiveness to IL-4 and IL-13 did not have a detrimental effect during *L. donovani* infection. Here we expand on these findings and show that CD4+ T cell-(Lckcre), as well as pan T cell-(iLckcre) specific IL-4Rα deficient mice, on a BALB/c background, unlike global IL-4Rα deficient mice, are also not adversely affected in terms of resistance to primary infection with *L. donovani*. Our analysis suggested only a transient and tissue specific impact on disease course due to lack of IL-4Rα on T cells, limited to a reduced hepatic parasite burden at day 30 post-infection. Consequently, the protective role(s) demonstrated for IL-4 and IL-13 during *L. donovani* infection are mediated by IL-4Rα-responsive cell(s) other than macrophages, neutrophils and T cells.

Keywords: *Leishmania donovani*, IL-4Rα, IL-4, T cell, mice

### INTRODUCTION

Infection with the parasite Leishmania donovani causes visceral disease and can be fatal if it is not treated. Although there are major campaigns aimed at eliminating this disease e.g., World Health Organization 2020 roadmap, it still remains a serious neglected tropical disease (1) (https:// www.who.int/leishmaniasis/en/), with no effective vaccine currently available (2). Successful pathogenesis is dependent on parasite survival in the host, a process mediated by a complex interplay of host factors. An in-depth understanding on the contribution of these factors to disease is therefore necessary to inform the development of novel or adjunct host-directed therapies (3, 4).

Earlier studies in this context revealed that the IFN-γ/IL-4 paradigm of resistance and susceptibility to intracellular infection, as defined for species causing cutaneous leishmaniasis (CL), does not apply holistically to species causing visceral leishmaniasis (VL). As with CL, protective immunity against this parasite relies on a Th1 response, which requires IL-12 production, and culminates in IFN-γ release (5, 6). In target tissues such as the liver, infection results in granuloma formation around infected macrophages (Kupffer cells) and eventual parasite death, primarily via the action of reactive nitrogen and oxygen intermediates (7, 8). However, unlike CL, a dominant inhibitory role for type 2 cytokines is less clear in murine models of VL (9). In asymptomatic and cured VL patients (10–12), both IFN-γ and IL-4-producing T cells have been identified and in the murine model of VL, protection is related to higher frequencies of cytokine-producing cells rather than altering the IFN-γ/IL-4 balance (13). In contrast, both human (12, 13) and murine (14) VL studies show that IL-10 is more important than IL-4 in immune suppression and parasite persistence.

Rather than being a detrimental cytokine for host protection, the evidence tends to suggest that type 2 immune responses may actually contribute to control of VL. Accordingly, our previous studies utilizing gene-deficient mice have identified protective roles for IL-4, IL-13, and IL-4Rα signaling during primary L. donovani infection (15–17). Control of parasite growth within the liver depends on the ability of Kupffer cells to clear parasites inside mature granulomas (15), a mechanism which requires T cell-derived IFN-γ (18) and the coordinated activity of macrophages which migrate toward the infected area. Enhanced susceptibility of IL-4−/−, IL-13−/−, and IL-4Rα −/− mice to L. donovani infection was associated with a reduction in type 1 responses and retarded granuloma maturation so that fewer mature or sterile granulomas were present (15, 16, 19). In line with these observations, a recent study indicated that IL-10, and not IL-4, was responsible for manipulating monocytes/macrophages in VL infection (20). In addition to playing significant roles in controlling primary infection with L. donovani, IL-4, and IL-13 have also been associated with the successful outcome of sodium stibogluconate (SSG) treatment and vaccination with recombinant hydrophilic acylated surface protein (HASP)B-1 (15, 17, 19). While these studies added value to our understanding of the contribution of IL-4/IL-13 in hostprotection against VL, they did not provide information on which cells were targeted by these cytokines via the IL-4Rα, and are therefore critical for protective immunity. Indeed, both IL-4 and IL-13 are pleiotropic cytokines and numerous cell types of both the innate and adaptive immune system produce and respond to these cytokines (21). In this regard, studies in celltype specific IL-4Rα-deficient mice during CL has revealed a hierarchical interaction between the IL-4Rα chain and its ligands on different immune cells. To illustrate this, IL-4Rα signaling via DCs to produce IL-12 plays a protective role during cutaneous infection with Leishmania major (22), while IL-4Rα signaling via T cells (23) and Th2 induction, via macrophages and alternative activation (24), and via B cells and IL-4 production (25), all promote disease progression.

To further dissect the cell-specific requirements of IL-4/IL-13 signals on immune cells in VL, we used conditional cell-specific IL-4Rα deficient BALB/c mice, generated by the cre/loxP recombination system, to demonstrate that macrophage/neutrophil-specific (LysM) IL-4Rα signaling was not necessary for an effective healing response during VL, nor did it influence the outcome of SSG chemotherapy (16). Other possible target cells for IL-4 during VL may include dendritic cells (DC) (26, 27) and B cells (28) but more particularly CD4+ (26, 29) and/or CD8+ (30) T cells, whose active involvement has been shown not only to be essential to control primary infection and granuloma formation but also for successful SSG chemotherapy and therapeutic vaccination (15, 31, 32). Consequently, in this study we generated CD4+ T cell-specific IL-4Rα −/− (LckcreIL-4Rα −/lox) mice (23) and iLckcreIL-4Rα −/lox mice that lack IL-4Rα on both CD4 and CD8 T cells (33) to determine the temporal role of IL-4 signaling via CD4+ and CD8+ T cells on the progression of VL infection. Unlike global IL-4Rα −/− mice infected with L. donovani that developed significantly higher parasite burdens than wild-type mice in this and previous studies (15), CD4+ T cell specific IL-4Rα −/− mice were by comparison resistant to infection. Indeed, at day 30 post-infection CD4+ T cell as well as pan T cell-specific IL-4Rα −/lox mice (iLckcreIL-4Rα −/lox) were more. resistant than their wild-type littermate controls to hepatic infection with L. donovani. Increased susceptibility in global IL-4Rα −/− mice was associated with a diminished type 1 response and increased IL-10 production while CD4+ T cell deficient IL-4Rα −/lox mice had comparable expression of IFN-γ on both CD4+ and CD8+ T cells and serum IL-10 levels similar to infected wild-type mice. Consequently, the protective effects of IL-4 during primary L. donovani infection are not mediated via direct effects on either CD4+ or CD8+ T cells, and IL-4 may even play a small regulatory role in these cells.

### MATERIALS AND METHODS

### Ethics Statement

Animal experiments and experimental procedures were carried out in line with UK Home Office regulations and the University of Strathclyde Animal Welfare and Ethical Review Board regulations under project license number: PPL 60/3525. BALB/c mice were all bred and maintained in the Biological Procedures Unit at the University of Strathclyde, Glasgow and experimental design and reporting adhere to the ARRIVE guidelines.

### Animals and Parasites

LckcreIL-4Rα −/lox BALB/c mice were created as described (23). Briefly, LckcreIL-4Rα −/lox mice were generated by inter-crossing IL-4Rα lox/lox BALB/c mice (34) with IL-4Rα −/− BALB/c mice (35) and BALB/c mice expressing the Cre-recombinase under the control of Lck locus, a T cell specific promoter (36) to generate hemizygous LckcreIL-4Rα −/lox mice. The efficiency of the deletion was characterized (23) whereby LckcreIL-4Rα −/lox BALB/c mice have IL-4Rα selectively deleted from CD4+ T cells (CD4+ T cell specific IL-4Rα deficient mice). As these mice have variable and incomplete deletion in CD8+ T cells (28), mice lacking the IL-4Rα on all T cells (iLckcreIL-4Rα −/lox mice) were produced as described (33). The iLckcre construct was designed with a nuclear localization signal and an eukaryotic translation start site at the 5' end of the Cre-recombinase, which meant that the insert integrated downstream from the Lck proximal promoter (33, 37). Non-T cell populations such as B cells, macrophages and DC express IL-4Rα as normal. PCR genotyping studies were used to identify LckcreIL-4Rα −/lox mice. Lckcre negative IL-4Rα −/lox littermate BALB/c mice, which express similar levels of IL-4Rα as wild-type BALB/c mice, were used as controls to iLckcreIL-4Rα −/lox mice, herein referred to as wild-type (WT) littermate control.

Commercially obtained Golden Syrian hamsters (Mesocricetus auratus) were used for maintenance of L. donovani (Harlan Olac, Bicester, UK) and L. donovani strain MHOM/ET/67:LV82 was used in these studies (38). Mice, age and sex-matched, were coded and randomized before injection to avoid bias. The mice were infected (day 0) by intravenous injection into the tail vein without using an anesthetic with 1–2 × 107 L. donovani amastigotes (16). Depending on the experiment, mice were killed (n = 4/5 per group) at days 14 or 15, 30, and 56 or 60 post-infection (p.i.).

### *In vivo* Studies

Liver, spleen, and bone marrow impression smears from each mouse were prepared on a glass microscope slide at sacrifice. The slides were fixed in methanol for 2 min, stained with a 10% Giemsa solution (BDH, VWR International Ltd, UK) for 20 min, washed in tap water and allowed to dry. The number of parasites /1,000 host nuclei for each sample was determined at ×1,000 magnification. Blood was collected from each mouse at sacrifice and left to clot. The resulting serum was harvested, stored at −20◦C, and then used in assays to determine specific antibody titers and cytokine levels.

### Spleen Cell Proliferation Assays

Mouse spleens were removed aseptically at sacrifice, and processed as described previously (16). Splenocytes, seeded at 5 × 105 cells/well, were incubated at 37◦C and 5% CO2/95% air for 72 h with medium alone (unstimulated controls), 25µg/ml L. donovani promastigote soluble antigen (SAG) or 10µg/ml ConA (Sigma-Aldrich, Poole, UK). The plates were then stored at −20◦C and cell supernatants were used in cytokine/nitrite assays.

### Cytokine Production

Enzyme-linked immunosorbent assays (ELISA) was used to determine IFN-γ levels in splenocytes supernatants from lymphocyte proliferation assays and serum samples using the method described (19). A volume of 50 µl/well of 1µg/ml w/v of the appropriate purified anti-mouse capture antibody [IL-10 JES5-2AS and IFNγ R4-6A2 (PharMingen, supplied by Insight Biotechnology, Wembley, UK) or IL-13 38213 (R&D Systems Europe Ltd, Abingdon, UK)] and 100 µl/well of the appropriate biotinylated rat anti-mouse monoclonal antibody at 2µg/ml [IL-10 SXC-1 and IFNγ XMG1-2 (PharMingen, supplied by Insight Biotechnology, Wembley, UK) or IL-13 (R&D Systems Europe Ltd, Abingdon, UK)] was used in assays. The absorbance of the samples was read at 405 nm using a SOFTmax Pro (Molecular devices, California, USA) and cytokine concentrations (ng/ml) in samples was determined by linear regression using the standard values. In all cases the correlation coefficient was 0.970 or better.

### Specific Antibody Titers

Specific IgG1 and IgG2a titers were determined for serum samples using the method described previously (16). Plates were coated with 100 µl/well of 10µg/ml L. donovani SAG and probed using anti-mouse IgG1 (1:20,00 dilution) or IgG2a (1:10,000 dilution) HRP conjugates (Southern Biotechnology, supplied by Cambridge BioScience Ltd, Cambridge, UK). The absorbance of samples was determined at 450 nm using a SOFTmax Pro (Molecular devices, California, USA) and end point titers, defined as the last dilution to give an absorbance above background levels, was determined.

### Determination of IgE Production

A 96 well microtiter plate (Greiner Bio-One, Germany) was coated with 50 µl/well of 1µg/ml IgE purified anti-mouse capture antibody (clone R35-72 obtained from PharMingen and supplied by Insight Biotechnology, Wembley, UK) diluted in PBS pH 9.0 and incubated at 4 ◦C overnight. The plate was washed three times with PBS supplemented with 0.05% v/v Tween 20 (Sigma-Aldrich, Poole, UK), dried and blocked with 10% v/v FCS/PBS. Following addition of 200 µl/well the plate was incubated at 37◦C for 1 h. The plate was washed 3 times, dried and serum samples diluted at 1/50 were serially diluted in 10% v/v FCS/PBS along the plate. The plate was incubated at 37◦C for 2 h. Following incubation, the plate was washed 4 times and 100 µl/well of a biotinylated rat anti-mouse IgE monoclonal antibody (clone R35-118 obtained from PharMingen and supplied by Insight Biotechnology, Wembley, UK) diluted 1/5,000 in 10% v/v FCS/PBS was added to the plate. The plate was then incubated at 37◦C for 1 h. The plate was washed 5 times and 100 µl/well of streptavidin alkaline phosphatase (obtained from PharMingen and supplied by Insight Biotechnology, Wembley, UK) diluted at 1/2,000 in 10% v/v FCS/PBS was added. The plate was incubated at 37◦C for 45 min. The plate was washed 6 times and 100 µl/well of substrate (1 mg/ml p-nitrophenylphosphate (Sigma-Aldrich, Poole, UK) in 0.1 M glycine buffer, pH 10.4) was added. The plate was incubated in the dark at 37◦C until an appropriate yellow color developed. The absorbance of the samples at 405 nm was determined using a SOFTmax Pro (Molecular devices, California, USA) and the endpoint dilution determined as described above.

### Histology

Sections of liver were removed at sacrifice, fixed in neutral buffered formalin, and then processed for staining with Haematoxylin and Eosin (Fisher Scientific, Loughborough, UK). Granulomas were scored on their level of maturity based on the following criteria; infected Kupffer cells (parasitized macrophages), immature (developing granuloma consisting of CD4+ and CD8+ T cells and monocytes surrounding infected Kupffer cells), mature (more developed than immature) or sterile (parasite free granuloma) (39).

### Flow Cytometry

The percentage of CD4+IFNγ + and CD8+IFNγ + cells present in the spleen of mice was determined by flow cytometry. Single cell suspensions were prepared from the spleens of uninfected and infected mice in complete medium [RPMI 1640 (Lonza, Belgium) supplemented with 1% v/v of 2 mM L-glutamine solution and 1% v/v of 100 IU/ml Penicillin-100µg/ml Streptomycin (PAA Laboratories, GmbH, Austria), and 10% v/v FCS (Sigma Aldrich, Poole, UK)]. Cell suspensions were pelleted by centrifugation at 1,000 rpm, 4◦C for 5 min and the pellets were resuspended in 3 ml of erythrocyte lysing solution (0.007 M ammonium chloride, 0.085 M Tris, pH 7.2). Cell suspensions were incubated at 37◦C for 5 min, washed three times in complete medium, and then resuspended at a concentration of 1 × 106 /ml. The cells were incubated for 4 h with 2 µl/ml Brefeldin A (BD Biosciences, UK), 500 ng/ml ionomycin and 50 ng/ml phorbol 12-mysristate 13 acetate (Sigma Aldrich, Poole, UK) at 37◦C and 5% CO2. The cells were then washed with FACS buffer (5% w/v Bovine serum albumin, Fraction V (Sigma Aldrich, Poole, UK), 2 mM EDTA in PBS pH 7.4) for 5 min at 500 g, and then resuspended in FACS buffer containing 1µg/ml purified rat anti-mouse CD16/CD32 (FCγ111/11 receptor) monoclonal antibody (BD Biosciences, Oxford, UK) and incubated for 20 min. The cells were washed as before and stained with the appropriate anti-mouse antibody diluted in FACS buffer (0.5µg/ml PerCP-labeled anti-CD4+ or 1µg/ml APC-labeled anti-CD8+ or 0.5µg/ml PerCP and 1µg/ml APC-labeled IgG isotype controls, BD Biosciences, Oxford, UK) in the dark for 60 min at 4◦C. Cells were washed three times and then incubated with a 10% v/v cell lysing solution diluted in distilled water (BD Biosciences, Oxford, UK) for 10 min in the dark. The cells were washed again and incubated for a further 10 min in the dark with a 10% v/v permeabilizing solution diluted in distilled water (BD Biosciences, Oxford UK). Following this incubation, cells were washed and resuspended in 2µg/ml purified rat anti-mouse CD16/CD32 (FCγ111/11 receptor) monoclonal antibody (BD Biosciences, Oxford, UK) diluted in FACS buffer. After a 10 min incubation, PE-labeled anti-mouse IFNγ (final concentration 2µg/ml) and PE-labeled IgG isotype control (final concentration 2µg/ml, BD Biosciences, Oxford, UK) diluted in 10% v/v permeabilizing solution was added to the appropriate sample and the cells were incubated for 60 min. The cells were washed once more, and then resuspended in 400 µl of a 10% v/v cell fix solution diluted in distilled water (BD Biosciences, Oxford, UK). The number of positive staining cells for a specific marker was determined using a FACSCantoTM (BD Biosciences, Oxford, UK). Color compensation using BDTM Compbeads and the antibodies used to stain cells was carried out before cell data was collected based on forward and side scatter using FACsDivaTM software.

### Statistical Analysis of Data

Downstream data analysis was performed blind to treatment group and experiments were repeated at least twice when significant differences between treatments were obtained. Parasite burden from in vivo experiments were analyzed using a one-way ANOVA using log10 transformed data, followed by a Fisher's PLSD test to analyze differences between treatments using Statview R version 5.0.1 software package. Significant

days 30 and 56 post-infection with 1–2 × 107 *L. donovani* amastigotes in the liver (A), spleen (B), and bone marrow (C) by LDU. Representative data from one of three experiments performed (*n* = 5/group). \**p* < 0.05, \*\**p* < 0.01, \*\*\**p* < 0.001 compared to WT control (IL-4Rα −/lox), #*p* < 0.05 LckcreIL-4Rα −/lox compared to IL-4Rα −/−.

differences between treatments for cytokine and flow cytometry data were identified using a Kruskal Wallis test followed by a Tukey test. Granuloma maturation data was analyzed using a χ 2 test and the mean % granulomas in the Kupffer cell, immature, mature and sterile categories. Data was analyzed using Minitab ExpressTM version 1.51 software package and a p < 0.05 was considered significant.

### RESULTS

### A Protective Role for IL-4 During *L. donovani* Infection Involves Cell Targets Other Than CD4+ T Cells

CD4+ T cell-specific IL-4Rα deficient (LckcreIL-4Rα −/lox) and wild-type littermate control (IL-4Rα −/lox) and global IL-4Rα −/− BALB/c mice were infected with L. donovani on day 0 and parasite burdens in the spleen, liver, and bone marrow were

FIGURE 2 | CD4+ T cell specific IL-4Rα −/lox (LckcreIL-4Rα −/lox), wild-type control (IL-4Rα −/lox), and global IL-4Rα −/− BALB/c mice were infected with *L. donovani* on day 0 post-infection and sacrificed at days 30 (A) and 56 (B) post-infection. At each time-point, sections of liver were removed, processed, and stained with haematoxylin and eosin to enable scoring of hepatic liver granulomas. Representative data from one of two experiments performed (*n* = 5/group). On day 30 (A) the distribution was significantly different for control and global IL-4Rα −/− mice (χ 2 = 44; *p* < 0.00001) and CD4+ T cell specific IL-4Rα −/lox (LckcreIL-4Rα −/lox) and global IL-4Rα −/− mice (χ 2 = 63; *p* < 0.00001). On day 56 (B) the distribution was significantly different for control and global IL-4Rα −/− mice (χ 2 = 46; *p* < 0.00001) and CD4+ T cell specific IL-4Rα −/lox (LckcreIL-4Rα −/lox) and global IL-4Rα −/− mice (χ 2 = 30; *p* < 0.00001).

determined, on day 14/15, 30, and 56/60 p.i. All three mouse strains had similar parasite burdens in the spleen, liver, and bone marrow at early times p.i. (day 14 or 15; **Table S1**), indicating that the inability to signal through IL-4Rα on CD4+ T cells did not interfere with establishment and early control of infection in the spleen, liver, and bone marrow.

At day 30 p.i., CD4+ T cell IL-4Rα deficient mice and littermate controls had comparable parasite burdens in the spleen and bone marrow. However, CD4+ T cell IL-4Rα deficient mice had significantly lower liver parasite burdens than WT littermate control and global IL-4Rα −/− BALB/c mice (**Figure 1**) at this time. This effect was transient however, and by day 56 parasite burdens in all three sites was comparable between WT and CD4+ T cell-specific IL-4Rα deficient mice. In contrast, and as expected (15), global IL-4Rα −/− mice were more susceptible to L. donovani infection and this was exhibited by significantly higher parasite burden at later time points in all target tissues (**Figure 1**). Thus, selective deficiency of IL-4Rα expression in CD4+ T cells had a temporary protective effect, which was only expressed in the liver, but did not alter the overall susceptibility to VL.

### IL-4 Signaling Via CD4+ T Cells Is Not a Requirement for Effective Hepatic Granuloma Formation Following *L. donovani* Infection

Granuloma formation in livers of L. donovani infected mice was assessed at days 15, 30, and 56 post-infection in CD4+ T cell specific IL-4Rα deficient (LckcreIL-4Rα −/lox), wild-type littermate control (IL-4Rα −/lox) and global IL-4Rα −/− BALB/c mice. At day 15 post-infection, although granuloma development was evident, the granulomas were at an immature stage and, as such, no significant differences between groups were observed (**Table S2**). At day 30 post-infection, however, CD4+ T cellspecific IL-4Rα deficient mice and wild-type controls showed evidence of granuloma maturation, as defined by an increased number of mature and sterile granulomas, compared with global IL-4Rα −/− mice (**Figure 2A**). Similarly, at day 56, the frequency of mature and sterile granulomas had increased and was similar in CD4+ T cell-specific IL-4Rα deficient mice and wild-type control mice, reflecting a similar ability to control liver parasite burdens by this time point (**Figure 2B**). Granuloma maturation was significantly retarded in global IL-4Rα −/− but not CD4 T cell-specific compared with WT mice at both time points (p < 0.00001). Representative photomicrographs show granuloma formation in each group at days 15 (**Figure S1**), 30 (**Figures 3A–C**) and day 56 (**Figures 3D–F**) post-infection.

### Th2/type 2 Immune Responses Are Unchanged in CD4 T Cell-Specific IL-4Ra Deficient Mice in Response to *L. donovani*-induced VL

An inability of global IL-4Rα −/− mice to control parasite burdens or develop a mature and effective granulomatous response has previously been associated with a down-regulation in serum IFN-γ production (15). In the present study we therefore measured production of IFN-γ by antigen-stimulated spleen

cells and the frequency of CD4+IFN-γ + and CD8+IFN-γ + splenocytes of each mouse group after stimulation with PMA and ionomycin, as a measure of commitment to cytokine production during infection. On day 30 post-infection, we found no significant difference in the amount of IFN-γ [ng/ml] produced by antigen-stimulated splenocytes from any group of mice, WT controls (0.207 ± 0.030), CD4+ T cell specific IL-4Rα deficient mice (0.331 ± 0.049), global IL-4Rα −/− mice (0.482 ± 0.076). However, using flow cytometry to examine intracellular cytokine production at this time revealed that the percentages of CD4+IFN-γ + splenocytes (**Figure 4A**) and CD8+/IFN-γ + splenocytes (**Figure 4B**) in global IL-4Rα −/− mice were significantly lower (CD4+IFN-γ +, p < 0.05; CD8+IFN-γ +, p < 0.02) than observed in WT controls while the frequency of CD8+/IFN-γ + splenocytes in global IL-4Rα −/− mice was also reduced compared with CD4+ T cell specific IL-4Rα −/− mice (p < 0.01, **Figure 4B**). This pattern was repeated on day 60 post-infection, (e.g., p < 0.0001 for global IL-4Rα −/− mice vs. CD4+ T cell-specific IL-4Rα-deficient mice). There was no significant difference measured in the frequency of CD4+IFN-γ + and CD8+IFN-γ + cells between CD4+ T cell specific IL-4Rα deficient mice and wild-type controls at all time

points. Together, these data indicate that IFN-γ-responses in LckcreIL-4Rα −/lox mice developed fully in the absence of IL-4Rα + signaling on CD4+ T cells when compared with wildtype mice over the course of infection. Moreover, enhanced susceptibility to L. donovani infection in global IL-4Rα −/− mice is associated with reduced IFN-γ secretion by CD4+ and CD8+ T cells.

IL-10 levels are associated with susceptibility to L. donovani infection (14), thus, IL-10 levels were measured in cell supernatants of antigen-stimulated spleen cells and in the serum from CD4+ T cell specific IL-4Rα deficient mice, wildtype control mice and global IL-4Rα −/− mice at days 15, 30, and 56 post-infection. There was no significant difference in the amount of IL-10 [ng/ml] produced by antigen-stimulated cells from any group of mice at day 30, WT controls (0.281 ± 0.075), CD4+ T cell specific IL-4Rα deficient mice (0.231 ± 0.026), and global IL-4Rα −/− mice (0.230 ± 0.013). In addition, while similar levels of IL-10 were present in the serum of all three groups of mice at day 15 p.i, by day 30 p.i significantly lower concentrations of IL-10 were present in the serum of wild-type control (p < 0.01) and CD4+ T cell specific IL-4Rα deficient mice (p < 0.01) compared with global IL-4Rα −/− mice (**Figure 5**). This pattern was repeated on day 56 post-infection. This data suggests that the relative resistance observed in CD4+ T cell specific IL-4Rα deficient and littermate control mice, in comparison to highly susceptible global IL-4Rα −/− mice, is associated with comparatively limited IL-10 production.

### CD4+ T Cell Specific IL-4Rα-deficient Mice Develop *L. donovani*-specific IgG1 and IgE Antibody Responses Similar to WT Control Mice

L. donovani-specific IgG1 and IgG2a levels and total serum IgE levels were measured in CD4+ T cell specific IL-4Rα −/− mice, wild-type control and global IL-4Rα −/− mice at days 15, 30, and 56 post-infection. There was no difference in specific IgG1 or IgG2a in the three groups of mice at day 14 post-infection (**Figures 6A,B**). However at day 30 p.i., global IL-4Rα −/− produced significantly lower levels (p < 0.0.01) of antigenspecific IgG1 but significantly higher IgG2a levels compared with CD4+ T cell specific IL-4Rα deficient mice and wild-type controls. In contrast, IgG1 and IgG2a titers were similar in CD4+ T cell specific IL-4Rα deficient mice and wild-type controls (**Figures 6A,B**). On day 60 p.i., both global and CD4+ T cell specific IL-4Rα −deficient mice had significantly lower IgG1 titers compared with WT controls (p < 0.05). IgG2a titers were similar in all three groups of mice (**Figures 6A,B**). Comparison of the ratio of IgG2a:IgG1 showed that global IL-4Rα −/− mice had a higher ratio at days 30 (p < 0.01) and 56 (p < 0.001) p.i. compared with WT and CD4+ T cell specific IL-4Rα deficient mice (**Figure 6C**). Both CD4+ T cell specific IL-4Rα deficient mice and wild-type control mice produced comparable amounts of IgE on days 30 and 56 post-infection (**Figure 6D**). In contrast, no serum IgE was detected for global IL-4Rα −/− mice any day post-infection (**Figure 6D**). Based on this antibody data, it can be concluded that the inability to signal through the IL-4Rα on CD4+ T cells did not prevent a specific type 2 antibody response whereas the inability to class switch in the global IL-4Rα −/− mice resulted in a reduced type 2-antibody response.

### Pan T Cell-Specific IL-4Rα Deficient Mice Remain Comparatively Resistant to VL Compared With Mice Globally Deficient for IL-4/IL-13 Signaling

As CD4+ T cell-specific IL-4Rα-deficient mice remain positive for IL-4Rα expression on CD8 T cells, we could not rule out the possibility that IL-4Rα + CD8+ T cells in CD4+ T cell specific IL-4Rα − deficient mice could have contributed to infection control, especially since CD8+ T cells have been implicated in protective immunity to VL (15, 30). Thus, we sought to investigate this further by using pan T cell specific IL-4Rα-deficient mice (iLckcreIL-4Rα −/lox) which lack IL-4Rα signaling on CD4 T cells as well as CD8 and Foxp3 regulatory T cells (33). iLckcreIL-4Rα −/lox, IL-4Rα −/lox littermate control and global IL-4Rα −/− mice were infected with L. donovani and parasite burdens analyzed at day 30 p.i. in the spleen, liver, and bone-marrow. All groups of animals exhibited similar parasite numbers in the spleen and bone-marrow at day 30 p.i. However, pan T cell IL-4Rα −/lox exhibited disease-control in the liver compared with WT littermate controls (p < 0.01) and global IL-4Rα −/− mice (p < 0.01, **Table 1**), similar to but more pronounced than that seen in CD4+ T cell-specific IL-4Rα-deficient mice (**Figure 1A**).

Altogether, these data indicate that IL-4Rα signaling on T cells transiently exacerbates infection in the liver after infection with L. donovani. These results also reinforce the ability of IL-4/IL-13 to exhibit temporal and spatial regulation depending on celltype and host tissue involved during infection. However, given that IL-4/IL-13 signaling globally is protective in VL, our results clearly demonstrate that the T cell is ultimately not the IL-4Rα responsive target mediating this protection.

### DISCUSSION

While it is well-established that protective immunity against leishmaniasis, whether cutaneous or visceral, relies upon an IL-12-driven type 1 response and IFN-γ production, the contribution of IL-4, IL-13, and signaling via IL-4Rα to the outcome of Leishmania infection is very much parasitespecies dependent [reviewed (21, 40)]. Numerous studies have identified detrimental roles for IL-4, IL-13, and signaling via IL-4Rα, as well as IL-10, during cutaneous infection with L. major and Leishmania mexicana complex parasites. In contrast, experimental studies using gene-deficient mice in L. donovani infections have indicated that the control of not only primary infection, but also successful chemotherapy and successful vaccination is IL-4, IL-13, and IL-4Rα signaling-dependent (15– 17, 32). The absence of these cytokines, or the inability to signal via the IL-4Rα in BALB/c mice, results in a reduced IFN-γ response, severely limited granuloma development, enhanced IL-10 production and disease exacerbation. However, the mode of action of these cytokines continues to remain obscure as IL-4 and IL-13 are pleiotropic cytokines and many cell types are not only able to produce these cytokines but also are responsive to them via IL-4Rα (21). Consequently, the generation of spatial (cellspecific) IL-4Rα −/− mice has provided an invaluable resource to identify the specific role of IL-4/IL-13-responding cells in ongoing immune responses (15, 17, 19). A previous study by us using macrophage/neutrophil specific IL-4Rα-deficient BALB/c mice identified no role, whether protective or detrimental, for IL-4 or IL-13 signaling via these cells on the outcome of primary L. donovani infection (16). T cells are the primary source of IFN-γ production in acquired immunity against VL (30), and have been implicated as playing a major role in granuloma formation and resolution of infection in the murine model (39). Therefore, in the present study we utilized CD4+ T cellspecific IL-4Rα-deficient (LckcreIL-4Rα −/lox) mice (23), and as confirmation iLckcreIL-4Rα −/lox mice that lack IL-4Rα on both CD4+and CD8+ T cells (33) to determine if IL-4Rα signaling via T cells plays any important role in protection. The results reveal that IL-4Rα signaling via the CD4+ T cell is not a requirement for successful resolution of L. donovani infection, but the inability to signal via IL-4Rα did have a transient protective effect (day 30) on hepatic parasite burdens compared with wild-type counterparts. This hepatic protective effect was also observed in pan-T cell specific BALB/c mice i.e., iLckcreIL-4Rα −/lox BALB/c mice, confirming that this early response is mediated by IL-4Rα signaling on T cells. However, later postinfection, CD4+ T cell specific IL-4Rα-deficient mice and wildtype controls had equivalent parasite burdens in the spleen, liver and bone marrow as well as a similar hepatic granulomatous response. This is in contrast to global IL-4Rα −/− mice that developed significantly higher parasite burdens and abrogated hepatic granuloma maturation compared with CD4+ T cell specific IL-4Rα-deficient and wild-type mice in all tissue sites. Overall these data suggest that host-protection mediated by IL-4/IL-13 globally during L. donovani infection is not due to IL-4 acting on CD4+ T cell populations.

Earlier studies on L. donovani, not only in mice (9), but also humans (10–12), suggested that control of infection was independent of the differential production of type 1 and type 2 cytokines and murine studies indicate that protection is related to increasing the frequency of cytokine-producing cells rather than altering the IFN-γ/IL-4 balance (13). The results presented here confirm that resistance to L. donovani and induction

of effective granuloma production is dependent on successful generation of an IFN-γ response on both CD4+ and CD8+ T cells and while this is positively regulated by IL-4/IL-13 signaling on a global scale, it is not strictly dependent on CD4+ and CD8+ T cells signaling these cytokines. CD4+ T cells (26, 39) and CD8+ T cells (41) produce IFN-γ, which activates macrophages to produce antimicrobial reactive nitrogen and oxygen intermediates (7), and these are also important in driving granuloma maturation. The observations on comparative T cell IFN-γ expressing cell numbers from this study complement and reinforce our previous observation that global IL-4Rα −/− mice produce significantly lower levels of serum IFN-γ compared with wild-type mice following L. donovani infection, and contribute to their abrogated granuloma development (15).

In parallel, although IL-4 is important in proliferation and maintenance of CD4+ Th2 cells, it is not required for early production of Th2 cells (42, 43), and therefore the inability to signal via the IL-4Rα did not prevent the induction of antigenspecific Th2 cell response, shown by the similar antigen-specific IgG1 titers in L. donovani infected WT and CD4+ T cellspecific IL-4Rα-deficient (LckcreIL-4Rα −/lox) mice. This may not be unexpected as it has been suggested that antigen-specific Th2 cells is the default position for Th cells (41). The ability to produce antigen-specific IgG1 in the absence of IL-4Rα signaling has been reported in other studies (39, 40).

*Mice (n* = *5/treatment) were infected with L. donovani LV82 and parasite burdens were determined on day 30 post-infection.* \*\**p* < *0.001 compared to WT control,* \*\**p* < *0.01 compared to WT control, #p* < *0.01 iLckcreIL-4R*α −/*lox compared to IL-4R*α −/−*.*

Recently, a pivotal role for IL-4 and IL-4Rα-responsive B cells in the non-healing response of BALB/c mice infected with L. major has been demonstrated (25). Abrogation of IL-4Rα signaling on B cells in BALB/c mice (mb1creIL-4Rα −/lox) turned non-healer BALB/c mice into a healer phenotype concomitant with a switch from a predominately Th2 to a Th1 response. Regulatory B cells producing IL-10 have also been associated with non-healing L. major infection (44) although apparently not associated with IL-4 responsiveness. However, the role of B cells (as APCs or regulatory cells) and/or antibody in contributing to susceptibility to Leishmania infection appears to depend in large part upon the parasite species examined but also probably to a significant extent upon the host. B cells have been shown to play a role in VL, thus B cell-deficient C57BL/6 mice infected with L. donovani (45) and BALB/c mice infected with L. infantum (46) are relatively resistant to infection. Susceptibility to infection in B cell deficient C57BL/6 mice, unlike BALB/c mice is not dependent on antibody production, although antibodies are associated with protection in infected C57BL/6 mice as they prevent excessive pathology. Marginal zone B cells regulate antigen-specific CD8+ T cells responses (47), indicating that B cells may be protective via their ability to present antigen. In L. infantum-infected BALB/c mice, neither IL-10 production by B cells nor antigen presentation by B cells is involved in disease exacerbation. However, passive transfer experiments have shown that IgM and IgG, induced by polyclonal B cell activation during infection, promote parasite growth. It is possible that the role of B cells differs in the two mouse strains and is related to their inherent "cure" phenotype (C57BL/6) and "non-cure" phenotype in L. donovani (48). B cells have been observed to have cognate interactions with T cells in hepatic granulomas in BALB/c mice (28) and problems with antigen presentation to CD8+ T cells have been reported in L. donovani (30). There were clear differences in antibody responses between global IL-4Rα −/− mice and the other two strains, with differences in total IgE and antigen-specific IgG1 being the most marked. Production of these antibody classes by B cells is known to be controlled by IL-4/IL-13 and requires IL-4Rα signaling (49). The fact that CD4+ T cell specific IL-4Rα −/− mice can still produce IgE indicates that a comparative examination of L. donovani disease phenotypes in B cell specific IL-4Rα −/lox BALB/ c mice would be worthwhile.

Both human (12) and murine studies (14, 50) indicate that IL-10, rather than IL-4/IL-13 is the major immunosuppressive cytokine in VL. The source of this IL-10 is not traditional Th2 cells (13) but perhaps a regulatory T cell population (51), and different types of regulatory T cells have been identified in L. donovani, including CD4+CD25−FoxP3− T cells in humans (52), and CD4+CD25−FoxP3+ in mice (53) and humans (54). In addition, numerous non-T cell populations, including DCs (55) and NK cells (56) have been implicated as playing regulatory roles during VL. Thus, L. donovani infected IL-10 gene deficient mice demonstrated increased control over visceral infection and enhanced granuloma formation, whilst IL-10 transgenic mice developed a severe progressive disease (50, 57) clearly demonstrating that the presence of IL-10 can prevent efficient granuloma development (50). Indeed, in the present study, global IL-4Rα −/− mice had impaired hepatic granuloma development alongside elevated serum IL-10 levels compared with wildtype control mice and CD4+ T cell specific IL-4Rα −/− mice. Interestingly it has previously been shown that IL-4 inhibits IL-10 to promote IL-12 production in DCs in the presence of CpG or LPS (58), or Cryptococcus neoformans (59), while IL-13 can induce DC IL-12 production in vitro when used to prime DC prior to LPS stimulation (16). IL-4 treatment of BALB/c mice pre-T cell priming has previously been demonstrated to instruct DC to produce IL-12 and facilitate a protective Th1 response against L. major (60). In addition, deletion of IL-4Rα on DCs renders BALB/c mice hypersusceptible to L. major (22). DC IL-12 production in the early phase of L. donovani infection (61–64), in particular, has been identified as directing immune responses influencing granuloma formation during infection. Consequently, these observations, collectively, would clearly point to DCs as very probable targets of the IL-4/IL-13 induced protective response identified from our studies on IL-4−/−, IL-13−/−, and IL-4Rα −/− mice.

### CONCLUSION

A significant number of studies from our laboratories have identified protective roles for IL-4, IL-13 and IL-4Rα signaling, not merely during primary infection with L. donovani infection (15–17), but also for effective sodium stibogluconate chemotherapy (16, 19). In addition, there is a requirement on IL-4/IL-13 to instruct a protective type 1 response mediated by CD8+ T cells in HASPB-1 vaccination against L. donovani (65). Given the pleiotropic nature of the IL-4Rα, these IL-4Rαresponsive cells could of course be different populations in different tissue sites and could vary for different type of immune responses e.g., primary infection, response to chemotherapy or successful vaccination. In lieu of this dynamic regulation, as yet our studies have failed to identify the IL-4Rα-expressing cells mediating protection during VL. So far we have ruled out a protective role in VL for IL-4Rα responsiveness on neutrophils and macrophages in primary infection and chemotherapy, and herein T cells during primary infection. Interestingly, the primary source of IL-4 in HASPB-1 vaccination studies was defined as a CD11b+CD11clo phagocyte (65) and as this

source is clearly not macrophages/neutrophils (16), alternate phagocytes expressing CD11b may regulate host immunity to VL. A candidate phagocyte for this role could be DCs given the "IL-4 instruction theory," defined as the ability of early IL-4, signaling via the IL-4Rα on DCs, to instruct early IL-12 production to promote Th1-responses (22, 60). We are currently investigating the exact mechanism to explain how this works as well as generating a variety of additional IL-4Rα cell specific knockouts to allow further dissection of the immunological mechanisms responsible for IL-4Rα mediated protection against VL. Ultimately identifying the IL-4Rα responsive host cells mediating protection will have significant implications in the rational design of new host-directed therapeutic strategies.

### DATA AVAILABILITY

The datasets generated for this study are available on request to the corresponding author.

### AUTHOR CONTRIBUTIONS

JA, FB, and PK: conceptualization. EM, KC, TM, and RH: methodology and investigation. EM, KC, JA, and RH: writing,

### REFERENCES

review, and editing of manuscript. FB: cell-specific genedeficient mice.

### FUNDING

This work was supported by grants from the Wellcome Trust, No. 065308 (PK, FB, JA), a Royal Society (U.K.) National Research Foundation (S.A.) Capacity Development Award (JA, FB), National Research Foundation (NRF) and the South African Research Chair Initiative (SARCHi) (FB), and National Research Foundation, South Africa (RH).

### ACKNOWLEDGMENTS

The authors would like to thank the staff within the BPU for their technical assistance and the genotyping and research animal facility and staff at University of Cape Town, South Africa.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2019.01957/full#supplementary-material

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 McFarlane, Mokgethi, Kaye, Hurdayal, Brombacher, Alexander and Carter. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Parasite-Produced MIF Cytokine: Role in Immune Evasion, Invasion, and Pathogenesis

#### Swagata Ghosh1 , Nona Jiang2 , Laura Farr 1 , Renay Ngobeni 3 and Shannon Moonah1 \*

1 Division of Infectious Diseases and International Health, Department of Medicine, University of Virginia, Charlottesville, VA, United States, 2 Department of Medicine, Yale University, New Haven, CT, United States, 3 Department of Environmental, Water, and Earth Sciences, Tshwane University of Technology, Pretoria, South Africa

Protozoan parasites represent a major threat to health and contribute significantly to morbidity and mortality worldwide, especially in developing countries. This is further compounded by lack of effective vaccines, drug resistance and toxicity associated with current therapies. Multiple protozoans, including Plasmodium, Entamoeba, Toxoplasma, and Leishmania produce homologs of the cytokine MIF. These parasite MIF homologs are capable of altering the host immune response during infection, and play a role in immune evasion, invasion and pathogenesis. This minireview outlines well-established and emerging literature on the role of parasite MIF homologs in disease, and their potential as targets for therapeutic and preventive interventions.

#### Edited by:

Jude Ezeh Uzonna, University of Manitoba, Canada

#### Reviewed by:

Nahed Ismail, University of Illinois at Chicago, United States Theo Araújo-Santos, Federal University of Western Bahia, Brazil Michael Makokha Opata, University of Texas Medical Branch at Galveston, United States

> \*Correspondence: Shannon Moonah<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Microbial Immunology, a section of the journal Frontiers in Immunology

Received: 27 May 2019 Accepted: 07 August 2019 Published: 21 August 2019

#### Citation:

Ghosh S, Jiang N, Farr L, Ngobeni R and Moonah S (2019) Parasite-Produced MIF Cytokine: Role in Immune Evasion, Invasion, and Pathogenesis. Front. Immunol. 10:1995. doi: 10.3389/fimmu.2019.01995 Keywords: MIF, cytokine, protozoan parasites, host-parasite interaction, immune evasion, immunopathology, immunotherapeutic target

## INTRODUCTION

Protozoan parasites cause more than one million deaths annually. For example, Plasmodium falciparum, a protozoan parasite responsible for most human malaria, accounted for an estimated 200 million malaria cases and roughly 500,000 malaria deaths in 2015 (1, 2). The protozoan parasite Leishmania causes an estimated 50,000 deaths per annum through visceral leishmaniasis (3). Entamoeba histolytica is a protozoan parasite that causes colitis (inflammatory diarrhea). Millions of people are infected with E. histolytica, making amebic colitis a leading cause of severe diarrhea, estimated to kill more than 50,000–100,000 people each year (4–6). The protozoan parasite Toxoplasma gondii, which affects up to a third of the world's population, is adapted to survive and abide chronically in its host (7). The threats posed by protozoan parasites are further compounded by lack of any effective parasite vaccine, emerging drug resistance, drug toxicity, poor efficacy, and limited antimicrobial options (5, 8, 9). Therefore, identifying novel targets for therapeutic intervention and vaccine prevention is urgently needed. Pathogenic protozoans produce virulence factors that enable immune response evasion and host invasion which promote their transmission and ability to cause human disease (10). Targeting these virulence factors required to cause host damage and disease might successfully treat and prevent these infectious diseases.

The pathogenesis of protozoan diseases is highly variable, and is often influenced by individual life cycles and immunologic consequences of infection. The complicated life cycle of Plasmodium begins when an infected female anopheles mosquito injects sporozoites into the bloodstream of a human during a blood meal, which travel to the liver, before emerging to release merozoites into the bloodstream. These merozoites invade and multiply within erythrocytes to rupture, releasing more merozoites, and continually perpetuating invasion by the parasite. Plasmodium promotes

**58**

its survival by avoiding excessive exposure to the immune system by infecting hepatocytes and erythrocytes. Clinical symptoms are associated with the rupture of infected erythrocytes and the release of malarial toxins, and include fever, severe hemolytic anemia and other systemic features. Merozoites also develop into sexual forms known as gametocytes, which are ingested during mosquito bites to continue the life cycle (11). Similarly, Leishmania is also a vector-borne protozoan parasite, that is transmitted when Leishmania promastigotes are inoculated into the subdermis of the skin by the bite of an infected female phlebotomine sand fly. Leishmania is rapidly phagocytized by neutrophils. Promastigotes within dead infected neutrophils are taken up by host macrophages, morphing into the amastigote form. Depending on the species, amastigotes replicate within the macrophage locally to form disfiguring skin ulcers (cutaneous leishmaniasis) or disseminate to the bone marrow, liver, and spleen (visceral leishmaniasis) which is fatal if untreated (12–15).

In contrast to these vector-borne infections, the transmission of the highly prevalent protozoa, Toxoplasma gondii, is fecal-oral, through the ingestion of the oocyst from material contaminated with feline feces or undercooked meat infected with tissue cysts. Following intestinal infection, tachyzoites form, and then disseminate to other tissues in the body including the brain, eye, muscle, liver, and placenta. Like Leishmania, Toxoplasma is able to infect phagocytes, which facilitates successful infection. Symptoms of primary infection include fever, adenopathy, headache, and myalgia. The stimulation of a robust immune response controls the acute infection, driving the parasite into a chronic, asymptomatic stage allowing Toxoplasma to survive as bradyzoites in cyst forms within multiple tissues capable of later reactivation (7). Infection with E. histolytica also begins with the ingestion of fecally contaminated food or water, but has a relatively simpler life cycle. E. histolytica exists as either infective cysts which are ingested or transforms into invasive trophozoites that penetrate the mucus layer of the large intestine to cause colitis leading to diarrhea, dysentery, and colonic ulceration. The trophozoites can also on occasion disseminate to cause extraintestinal disease, with a particular predilection for the liver leading to amebic liver abscess (16). Thus, in order to complete their life cycle, all of these protozoa must be able to invade and pass from host to host while avoiding clearance by the immune response. In this minireview, we describe how protozoa secrete a specific protein macrophage migration inhibitory factor to accomplish this task.

### MACROPHAGE MIGRATION INHIBITORY FACTOR

Macrophage migration inhibitory factor (MIF) was one of the first cytokines to be discovered over 50 years ago (17, 18). Since then, a significant amount of information has been accumulated regarding the role of MIF in normal physiology and pathology. MIF is a well-studied pleiotropic inflammatory protein, expressed by a variety of cells, and is a critical upstream mediator of innate immunity. While MIF's exact molecular mechanism is not fully understood, partial pathways of MIF signaling have been established. For example, secreted MIF binds to its receptor, CD74, on immune cells, activates the ERK1/2 and PI3K/Akt pathways, and modulates expression of various cytokines, e.g., TNF-α, IL-6, IL-8, and IL-12 (19). MIF may also bind to CXCR2 and CXCR4, which may be responsible for its chemotactic properties. In addition, MIF stimulates the production of matrix metalloproteinases (20). Therefore, it is not surprising that MIF plays an important role in immunity and that excess MIF expression has been linked to exaggerated inflammation and immunopathology in diseases such as rheumatoid arthritis, and inflammatory bowel disease (19, 21, 22).

The proinflammatory properties of MIF also make it a crucial mediator in the immune response against a wide variety of pathogens including parasites (23). In protozoan infection, host MIF play a key role in reducing parasite burden through stimulation of both innate and adaptive immune cells. Mechanistically, host MIF can stimulate nitric oxide production by macrophages and dendritic cells, which in turn eliminates parasites such as Leishmania, Toxoplasma, and Trypanosoma (13, 23, 24). MIF can also be harmful to the host. That is, MIF production has been linked to pathology during malaria and T. brucei infection, by promoting inflammationinduced tissue damage (21, 25, 26). The role of host MIF during parasite infections has been well-reviewed elsewhere (23, 24, 27).

Counterintuitively, many pathogenic protozoans, including Plasmodium, Entamoeba, Toxoplasma, and Leishmania, produce their own MIF cytokine. These secreted parasite-produced MIF are structurally similar to human MIF, bind the MIF receptor (CD74), and stimulate immune cells and epithelial cells to cause the release of cytokines such as TNF-α, IL-8, and IL-12 (28–35). While it seems counterintuitive for protozoans to secrete a proinflammatory cytokine, it appears they have an important role in the parasite life cycle. Here, we focus on MIF produced by medically important protozoans, highlighting the recent contributions that have improved our understanding of the role of protozoan MIF in immune evasion, invasion, and pathogenesis (**Figure 1**).

### IMMUNE EVASION

The host deploys a robust immune response to prevent parasite invasion, clear the infectious pathogen, and prevent re-infection. However, parasites have developed a remarkable number of mechanisms to evade these attacks (10). For example, Leishmania has developed ways to modify host cell signaling pathways, in order to survive and persist in host cells. Leishmania targets macrophages, which, interestingly, are the primary immune cells involved in the parasite's eradication (13). Leishmania major encodes two isoforms of MIF which facilitates its persistence in macrophages and contributes to its evasion from immune clearance. L. major MIF binds to CD74 on infected macrophages, activating the ERK1/2 pathway and preventing apoptosis of macrophages (35, 36). Infected macrophages then survive a sufficiently long enough time for the parasite to avoid excessive

exposure to the immune system and complete its infectious life cycle.

The lack of protective immunity against re-infection is one of the biggest problems in controlling the transmission of protozoan infections. An adequate amount of protective memory T-cells are needed to fight off re-infection (37). Recent research in parasite MIF has provided a mechanism by which parasites evade the immune response by interfering with the development of immunological memory during infection, allowing them to re-infect their host (28, 29, 35). Using mouse models, researchers found that the proinflammatory effects of both Plasmodium and Leishmania MIF can manipulate T-cell differentiation. Plasmodium MIF enhances the production of IFN-γ and IL-12 which reduces the anti-Plasmodium bloodstage CD4 T-cell response. Mice infected with MIF-deficient P. berghei had reduced levels of these cytokines. This reduced inflammatory state correlated with improved survival of CD4 T helper cells. As a result, mice were able to develop effective T-cell memory when infected with MIF-deficient parasites which provided a protective response against a subsequent P. berghei infection. Leishmania MIF cause T-cells to develop into exhausted PD-1+ short-lived effector cells with reduced IL-7R expression, which is needed to produce and maintain memory cells (28, 29, 35).  This MIF-induced lack of memory cells resulted in parasitic re-infection.

Recent clinical observations also support these findings. It was observed that in a cohort of children in an area endemic for amebiasis, those who lacked adequate amounts of antibodies against E. histolytica MIF were not protected from future infection (**Figure 2A**). The authors postulated that E. histolytica MIF might share similar properties to Plasmodium and Leishmania MIF. That is, E. histolytica MIF might also inhibit the development of sufficient amounts of memory cells. Thus, antibodies against E. histolytica MIF would block this effect resulting in adequate amounts of memory cells to protect against reinfection (32). Nevertheless, further studies are needed to confirm this theory. Also, the role of Toxoplasma MIF in immune evasion remains largely understudied.

### INVASION

Host tissue invasion by extracellular or intracellular protozoan parasites play an important role in the pathogenesis of disease. The extent of tissue invasion by extracellular parasites correlates with the degree of disease severity (16). For example, the depth of tissue invasion is associated with worse outcomes in clinicopathological studies of patients with severe amebic colitis (38, 39). On the other hand, obligate intracellular parasites invade host cells to complete their life cycle (13, 40). Several parasite factors are known to contribute to the invasion process. Recent studies have implicated parasite MIF proteins in facilitating invasion and dissemination of several protozoan parasites.

with permission.

The extracellular matrix (ECM) is a network of proteins that provides tissue support and represents a major physical barrier to the parasite invasion. Matrix metalloproteinases (MMPs) are enzymes primarily responsible for ECM breakdown (41). Protozoa infections trigger an inflammatory response which leads to MMP overexpression, resulting in ECM breakdown. ECM degradation facilitates cell movement, and allows immune cell infiltration at the site of infection for host defense (42). Parasites have developed mechanisms to exploit the activities of MMPs to promote their invasion. For example, MMPs play a critical role in E. histolytica invasion. MMP expression is increased in human amebic colitis and inhibition of MMP prevented E. histolytica invasion in a human colonic explant

EhMIF levels and the myeloperoxidase (MPO) marker of intestinal inflammation in persons with amebiasis (n = 35). Panels are reproduced from (17)

model (32, 43, 44). Recently, a causal relationship between E. histolytica-produced MIF and gut inflammation was established using cellular and mouse models of amebic colitis. In the same study, researchers found that E. histolytica MIF-induced inflammation resulted in increased MMP production (32). Therefore, E. histolytica parasites appear to produce MIF as a virulence factor to exploit the inflammatory response to promote tissue invasion.

As mentioned above, neutrophils are the first immune cells to reach the site of Leishmania infection after a sand fly bite, and their uptake by neutrophils followed by macrophage engulfment contributes to leishmanial parasites infectivity and assist in life cycle progression (12–14). Neutrophils are shortlived phagocytes that act as a "Trojan horse" used by Leishmania parasites to obtain entry into macrophages thereby avoiding cell activation (15). Whether there are leishmanial factors that drive this neutrophil infiltration remains largely unanswered. While host MIF exhibits chemokine-like activities through interactions with the chemokine receptors CXCR2 and CXCR4 (45, 46), protozoan MIF-CXCR2 and CXCR4 interactions remain unclear.

Similar to Leishmania, T. gondii induces immune cell infiltration and not only evades their killing, but also hitches a ride in these cells to spread infection (47, 48). T. gondii must cross the intestinal barrier for it to advance from the gut to sites of secondary infection, and tachyzoites are often found in neutrophils in the gut lumen. In vitro studies have indicated that T. gondii MIF stimulates the production of the potent chemoattractant IL-8 from human cells. In an attempt to explain why this would benefit the parasite, it has been suggested that MIF-induced IL-8 production leads to neutrophil recruitment. Infected neutrophils, which are incapable of clearing the parasite, serve as motile reservoirs for T. gondii infection, facilitating the transepithelial migration of the parasite (30, 48, 49). While plausible, additional studies are warranted to validate the role of Toxoplasma MIF in invasion.

### PATHOGENESIS

During protozoan infections, an unbalanced inflammatory reaction increases tissue destruction which leads to clinical disease. The inflammatory response is essential in that it provides protection against invading microbes. However, protozoan parasites have developed effective strategies to evade the immune response, avoid elimination, and persist in their host, which exacerbates the damage caused by the lingering inflammatory response to invading parasites (10). This is further compounded by the fact that these parasites secrete MIF cytokine that can directly drive inflammation.

Host cytokines released during Plasmodium infection contribute to severe malaria. For example, high TNF-α production is a strong predictor of severe malarial anemia and cerebral malaria in children (50, 51). P. falciparum MIF was shown to stimulate TNF-α secretion by immune cells in-vitro. Also, circulating serum P. falciparum MIF levels positively correlated with serum TNF-α levels in malaria patients, and higher P. falciparum MIF levels were observed in patients with severe malarial anemia and cerebral malaria (25, 28). These findings suggest that P. falciparum MIF is likely contributing to immunopathogenesis during malaria.

Neutrophil infiltration is a hallmark of amebic colitis. Neutrophils generate oxygen free radicals that are capable of killing the E. histolyica parasite. That said, E. histolytica has developed several strategies to counter and survive neutrophil killing (52–54). This results in an excessive and persistent neutrophil response in the gut that has been shown to be associated with the most severe forms of human amebiasis, which also carry high fatality (6, 55–57). E. histolyica MIF plays an essential role in neutrophil infiltration during infection. E. histolytica MIF was shown to stimulate IL-8 and the murine IL-8 homolog KC (potent neutrophil chemoattractants), resulting in neutrophil infiltration and tissue destruction in cellular and mouse models. A recent human study found that gut E. histolyica MIF levels correlated with intestinal inflammation severity [**Figure 2B**; (32, 33)].

Macrophages also play a crucial role in protozoan MIFinduced immunopathology. In vitro studies show that E. histolyica MIF directly enhances TNF-α and IL-6 production from macrophages (31). Both cytokines cause collateral tissue injury in amebic colitis and liver abscess (10, 58). In a mouse model of Leishmania infection, Leishmania MIF upregulated inflammatory and innate immune signaling in infected macrophages, such as CXCL1, TLR2, and TNF-α, when compared to MIF−/− strains (35). Taken together, this proinflammatory phenotype, extending the survival of infected macrophages, and defective adaptive immune response supports the contribution of Leishmania MIF to the chronic destructive inflammatory state observed in leishmaniasis.

### OTHER PROTOZOANS PRODUCING MIF

Other medically important protozoans include Trichomonas, Giardia, Trypanosoma, Acanthamoeba, and Naegleria. MIF orthologs have been discovered in Trichomonas and Giardia. The structure of Giardia MIF has been solved with a characterization similar to human MIF, but its role in infection is not wellunderstood (59). Surprisingly, MIF orthologs have not been characterized in Trypanosoma, Acanthamoeba, and Naegleria. However, incomplete genome assembly and annotation may limit in-silico analysis and explain why MIF has yet to be identified in these protozoans.

Inflammation is a critical component of tumor progression and many cancers, including prostate cancer, arise from sites

### REFERENCES

of infection and chronic inflammation (60, 61). Trichomonas vaginalis is a sexually transmitted parasite that can colonize the prostate in men. T. vaginalis also secretes MIF which has pro-inflammatory properties. In addition to stimulating the production of IL-8 and IL-6 cytokines, Trichomonas MIF binds to the human CD74 MIF receptor triggering the activation of the pro-proliferative ERK and P13K/Akt pathways in prostate epithelial cells. Trichomonas MIF-driven inflammation and cell proliferation, was linked to the promotion and progression of prostate cancer (62).

### CONCLUSION

Recent studies have made it increasingly clear that parasiteproduced MIF is a virulence factor that play a significant role in host-parasite interactions and contributes to pathogenesis. Despite these advances, key questions remain unanswered. Such as, can we translate these findings to provide beneficial interventions to patients infected with these pathogens? Do we know enough to intervene in a meaningful way? Protozoan MIF (P-MIF) appears to be a logical candidate for further evaluation as an effective immunotherapeutic target given the accumulation of data showing that: (i) infected persons naturally make antibodies against P-MIF, (ii) anti-P-MIF do not cross-react with host MIF, and (iii) neutralizing antibodies inhibit P-MIF activity and therefore prevent re-infection and reduce immunopathology.

### AUTHOR CONTRIBUTIONS

SG, NJ, LF, RN, and SM wrote different sections, edited, and reviewed the manuscript.

### FUNDING

This work was supported by National Institutes of Health (NIH) R01AI026649-27S1, K08AI119181, UVA seed grant, and the Robert Wood Johnson Foundation–Harold Amos Medical Faculty Development Program Award.

proteins attenuates its proinflammatory function. Sci Rep. (2018) 8:10241. doi: 10.1038/s41598-018-28625-1

factor from Giardia lamblia. J Struct Funct Genom. (2013) 14:47–57. doi: 10.1007/s10969-013-9155-9

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Ghosh, Jiang, Farr, Ngobeni and Moonah. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Computational Network Analysis Identifies Evolutionarily Conserved miRNA Gene Interactions Potentially Regulating Immune Response in Bovine Trypanosomosis

Olanrewaju B. Morenikeji1 , Megan E. Hawkes1 , André O. Hudson2 and Bolaji N. Thomas1 \*

1 Department of Biomedical Sciences, Rochester Institute of Technology, Rochester, NY, United States, 2 Thomas H. Gosnell School of Life Sciences, Rochester Institute of Technology, Rochester, NY, United States

### Edited by:

Jude Ezeh Uzonna, University of Manitoba, Canada

### Reviewed by:

Jata Shankar, Jaypee University of Information Technology, India Maryam Dadar, Razi Vaccine and Serum Research Institute, Iran

> \*Correspondence: Bolaji N. Thomas<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Microbial Immunology, a section of the journal Frontiers in Microbiology

Received: 28 June 2019 Accepted: 16 August 2019 Published: 28 August 2019

#### Citation:

Morenikeji OB, Hawkes ME, Hudson AO and Thomas BN (2019) Computational Network Analysis Identifies Evolutionarily Conserved miRNA Gene Interactions Potentially Regulating Immune Response in Bovine Trypanosomosis. Front. Microbiol. 10:2010. doi: 10.3389/fmicb.2019.02010 Bovine trypanosomosis is a devastating disease that causes huge economic loss to the global cattle industry on a yearly basis. Selection of accurate biomarkers are important in early disease diagnosis and treatment. Of late, micro-RNAs (miRNAs) are becoming the most useful biomarkers for both infectious and non-infectious diseases in humans, but this is not the case in animals. miRNAs are non-coding RNAs that regulate gene expression through binding to the 30 -, 50 -untranslated regions (UTR) or coding sequence (CDS) region of one or more target genes. The molecular identification of miRNAs that regulates the expression of immune genes responding to bovine trypanosomosis is poorly defined, as is the possibility that these miRNAs could serve as potential biomarkers for disease diagnosis and treatment currently unknown. To this end, we utilized in silico tools to elucidate conserved miRNAs regulating immune response genes during infection, in addition to cataloging significant genes. Based on the p value of 1.77E-32, we selected 25 significantly expressed immune genes. Using prediction analysis, we identified a total of 4,251 bovine miRNAs targeting these selected genes across the 30UTR, 50UTR and CDS regions. Thereafter, we identified candidate miRNAs based on the number of gene targets and their abundance at the three regions. In all, we found the top 13 miRNAs that are significantly conserved targeting 7 innate immune response genes, including bta-mir-2460, bta-mir-193a, bta-mir-2316, and btamir-2456. Our gene ontology analysis suggests that these miRNAs are involved in gene silencing, cellular protein modification process, RNA-induced silencing complex, regulation of humoral immune response mediated by circulating immunoglobulin and negative regulation of chronic inflammatory response, among others. In conclusion, this study identifies specific miRNAs that may be involved in the regulation of gene expression during bovine trypanosomosis. These miRNAs have the potential to be used as biomarkers in the animal and veterinary research community to facilitate the development of tools for early disease diagnosis/detection, drug targeting, and the rational design of drugs to facilitate disease treatment.

Keywords: cattle, miRNA-target, expression, trypanosomosis, evolution, immune response

### INTRODUCTION

fmicb-10-02010 August 27, 2019 Time: 17:48 # 2

Every year, the Cattle industry is faced with huge economic losses due to diseases such as trypanosomosis. Bovine trypanosomosis is endemic in sub-Saharan Africa and some parts of The Americas, causing losses of several billion dollars annually (Abdi et al., 2017; Odeniran and Ademola, 2018). The disease is associated with multifactorial complex phenotype, requiring an integrative biological approach to elucidate the molecular networks involved and to identify significant markers that may be useful for disease diagnosis and treatment (Noyes et al., 2011; Smetko et al., 2015; Kim et al., 2017). Recently, major research efforts are focused on utilizing microRNA (miRNA) as diagnostic biomarkers or therapeutic targets due to their involvement in various disease phenotypes and malignancies (Lawless et al., 2014; Scheel et al., 2017; Ammah et al., 2018; da Silveira et al., 2018). MicroRNAs are short, noncoding RNAs, which are capable of post-transcriptionally modifying gene expression, and form complexes with argonaute proteins that hybridize to the target mRNAs leading to the degradation of mRNA and negatively impact protein expression (Bushati and Cohen, 2007; Bartel, 2009; Meister, 2013; Huberdeau et al., 2017).

Post-transcriptional modification of gene expression is becoming a major focus in disease studies (Liu and Wang, 2019), with several reports showing miRNAs as important players in post-transcriptional regulation of both innate and adaptive immune gene expression (O'Connell et al., 2010; Sandhu et al., 2012; Kramer et al., 2015; Mehta and Baltimore, 2016; Pichulik et al., 2016; Kumar et al., 2018; Awuah et al., 2019). miRNAs have also been implicated in the regulation of immune functions in different cell types, for example neutrophil senescence and naïve mouse B cells have been reported to be regulated by repertoires of miRNAs (Ward et al., 2011; Bronevetsky et al., 2013; Aalaei-Andabili and Rezaei, 2016; Kangas et al., 2017). While there are numerous miRNAs abundantly expressed in bovine tissues and the entire genome, there are very few studies elucidating their regulatory role in bovine immunity (Coutinho et al., 2007; Jin et al., 2009; Xu et al., 2009; Hanif et al., 2018; Li et al., 2019). MicroRNAs therefore, will play important and unique roles during immune response, including modification of gene expression during bovine trypanosomosis. For instance, mir-2284 and mir-2285 families have been associated with TLR genes, thereby regulating non-specific immune responses through the production of proinflammatory cytokines, suggesting a role in bovine disease tolerance (Das et al., 2016). Similarly, mir-2404, mir-2285, and mir-6522 have been reported to regulate CD8 alpha chain and T-cell surface glycoprotein, thus playing significant role in development of T and B cell, proliferation, and inflammatory process (Mahjoub et al., 2015; Hanif et al., 2018). Studies have shown that miRNAs repress the target genes through the 30 untranslated region (30UTR) of mRNAs, therefore the miRNA binding site in this region seems to be well characterized (Buza et al., 2014; Liu and Wang, 2019). However, comparative genome analysis has shown that both coding domain sequence (CDS) and 30UTR sites are under selective pressure while many miRNA binding sites are conserved in both 30UTR and CDS (Hausser et al., 2013; Brummer and Hausser, 2014). In fact, many investigators are suggesting an alternative mode of gene regulation, whereby miRNAs anneal within the CDS, 5 0 - and/or 30 -UTR regions of their targets thereby regulating their translation.

Hence, there remains an important need to identify possible miRNA binding sites within the complete sequence of a gene (i.e., 50 -UTR, CDS and 30 -UTR), which can serve as potential biomarkers for disease diagnosis and treatment. To date, there has not been a published study identifying potential miRNAs as biomarkers in the entire sequence of immune genes responding during bovine trypanosomosis, demonstrating the uniqueness of this study. Notably, there are many miRNA databases and algorithms, which are available to investigate significant pathways and transcriptional mechanisms within various interaction and expression datasets (Xie et al., 2013; Li et al., 2014; Hanif et al., 2018). There are also proven in silico tools and algorithms available for miRNA target prediction and for dissecting miRNA function (Kogelman et al., 2014; Scheel et al., 2017; Hanif et al., 2018). A quantitative measurement of miRNA expression in conjunction with their target gene expression would be an important means to deconvolute diverse biogenesis pathways and immune system regulation during bovine trypanosomosis. To this end, we have employed a combination of computational tools to identify and elucidate conserved miRNAs, their target interaction(s), and regulatory function(s) during bovine trypanosomosis. This study, we believe, is a proof of concept with the potential to guide future studies aiming to identify attractive and putative miRNA targets for the rationale design and/or discovery of drugs to combat diseases, including potential biomarkers for disease diagnosis.

### MATERIALS AND METHODS

### Data Mining and Verification of Genes Associated With Bovine Trypanosomosis Pathway

In this study, we performed a data survey of genes that are reported to be significantly regulated during bovine trypanosomosis through publicly available databases. Briefly, we used LitInspector of Genomatix Literature Mining software (Version 3.10, Munich, Germany1 ) that uses proprietary literature data mining algorithms based on all available PubMed publications and their corresponding Medical Subject Headings (MeSH) (Berriz et al., 2003; Maier et al., 2005). This program gathers gene information and correlations with diseases and pathways from published literature. The gene sets are characterized based on annotation and literature including MeSH terms, gene ontology (GO) and pathways, as described (Scherf et al., 2005; Frisch et al., 2009). Out of the 3,738 disease terms found in the MeSH database, we selected only one

1https://www.genomatix.de/solutions/genomatix-software-suite.html

term relevant for our study, "trypanosomosis" (MeSH-Term ID: c03.752.300.900), using bovine as our reference species. The list of individual genes in association with bovine trypanosomosis as found in the scientific literature was filtered for significance to avoid random matches (Buza et al., 2014). Based on the parameters defined above, we selected a total of 25 genes that are significantly regulated and associated with our disease term based on the MeSH p-value (p = 1.77E-32). The 25 gene sets were then used as targets for further analysis (gene functions and references in **Supplementary Table S1**).

### Prediction and Extraction of Disease Associated Bovine miRNA

We searched for the identified 25 genes from our disease pathway and their locations within the bovine genome UMD 3.1 using the Ensembl BLAST/BLAT Genomic Sequence tool2 . Complete sequence of the 25 genes were retrieved with their accession number: CD86 (XM\_005201387), FcγR3A (NM\_001077402), CD1A (NM\_001105456), IL-6 (NM\_173923), IFN-G (NM\_174086), CD4 (NM\_001103225), CXCL-8 (NM\_173923), ICAM-1 (NM\_174348), CSF2 (NM\_174028), CD14 (XM\_005209429), TLR-4 (NM\_174198), LBP (NM\_001038674), IL-10 (NM\_174088), TLR-2 (NM\_174197), CCL2 (NM\_174006), MYD88 (NM\_001014382), TNF (NM\_173966), CD83(NM\_001046590), CD80 (NM\_001206439), IL-4 (NM\_173921), IL-18 (NM\_174091), LY96 (NM\_001046517), ITGAM (NM\_001039957), IL-12A (NM\_174355), and MAPKAPK3 (NM\_001034779). Thereafter, each gene was used as target to individually search the Bos taurus genome for possible miRNAs within their complete sequences (i.e., 30 -UTR, 50 -UTR and CDS) using miRWalk3 ; an online program which allows search for interactions between complete gene sequences and miRNAs using the TarPmiR algorithm. To avoid false positive miRNAs in our study, three other miRNAtarget prediction software (TargetScan4 , miRDB5 and miRBase6 ) were used to predict the miRNA and targets interaction for each gene. Only miRNAs that were confirmed in the three databases were included for further analysis. All miRNAs that were found from each gene target were partitioned into three regions; the 5 0 -UTR, CDS and 30 -UTR in order to examine the variation in miRNA abundance at each region of the targets. In order to identify common miRNAs among the three regions of each gene sequence, we sorted the matches using a web- based Venn diagram application7 .

### Network Analysis, Interaction of miRNA and Target Location of the Chromosome

Using the Ensembl BLAST/BLAT and the miRBase, we locate the position of each miRNA and their targets on the chromosomes within the bovine genome. All mature miRNAs sequences of our targets were retrieved; genes located on the same chromosome were pooled together for us to identify concomitant miRNAs coregulating one or more targets with the aid of an online Venn diagram8 . miRNA-target network was constructed with most significant genes in the disease pathway using miRNet; an online platform that integrate miRNAs, targets and their functions9 (Fan and Xia, 2018).

### Analysis of 7 Innate Immune Genes Associated With Bovine Trypanosomosis

We selected seven innate immune genes among the 25 that are responding in bovine trypanosomosis pathway for further analysis. Briefly, we used the accession numbers of TLR-4 (NM\_174198), ITGAM (NM\_001039957), ICAM-1 (NM\_174348), CD14 (XM\_005209429), TLR-2 (NM\_174197), LBP (NM\_001038674), and TNF-α (NM\_173966) to query the databases of miRWalk10 and miRBase11 in order to retrieve the mature and precursor miRNA sequences within the targets for further analysis. We selected only common miRNAs from the two databases as candidates for further analysis. Based on the number of targets by a miRNA, we selected the top 10 miRNAs regulating four to five targets. It is assumed that a miRNA with multiple targets is probably a key miRNA in the innate immune system under study (Newman and Hammond, 2010; Wang, 2010). We also compared the miRNA abundance at 50 -UTR, CDS and 30 -UTR of the selected top 10. This is also necessary assuming the regions with the most miRNAs will be more regulated and have greater impact on disease outcome.

### Evolutionary Trace and Phylogenetic Analysis of Conserved Bovine miRNA With Other Species

In order to determine the evolutionary trace and conservation of the identified bovine miRNA from the seven innate immune response genes, we selected the two miRNAs that showed the highest conservation from the 50 -UTR and 30 - UTR and 10 from the CDS being that it presented the most abundant miRNAs. Here, precursor miRNA sequences were retrieved from the miRBase database (see footnote 11); each bovine miRNA sequence was used for BLAST search with Ensembl BLAST/BLAT Genomic Sequence tool12. Homologous sequences from other species for each bovine miRNA were downloaded from the database for further analysis. Multiple sequence alignment (MAS) was performed using MEGA vs7 and Neighbor joining (NJ) phylogenetic tree was constructed with Cluster W to determine the nucleotide substitution and evolutionary divergence among the species. It is assumed that the most conserved miRNAs would be candidates for disease studies.

2http://www.ensembl.org/Tools/Blast/GenomicSeq

3http://mirwalk.umm.uni-heidelberg.de

4http://www.targetscan.org/vert\_72/

5http://mirdb.org/

6http://www.mirbase.org/index.shtml

7http://bioinfogp.cnb.csic.es/tools/venny/

8http://bioinformatics.psb.ugent.be/software/details/Venn-Diagrams 9https://www.mirnet.ca/miRNet/faces/upload/GeneUploadView.xhtml

10http://mirwalk.umm.uni-heidelberg.de/search\_genes/

11http://www.mirbase.org/index.shtml

12http://www.ensembl.org/Tools/Blast/GenomicSeq

### Gene Ontology and Functional Analysis of the Most Conserved Bovine miRNAs in Innate Immune System

We performed functional enrichment analysis using gene ontology (GO) term13 and DIANA tools14 for the topmost conserved miRNAs at the 30UTR, 50UTR and CDS of the 7 innate immune response genes to gain understanding of the possible biological processes and physiological pathways regulated by these miRNAs according to Paraskevopoulou et al. (2013). We used 13 topmost miRNAs from the 7 innate immune genes and 6 others that are significantly conserved from CXCL-8, TLR-4, and MAPKAPK3 (**Supplementary Table S2**), giving a total of 19 miRNAs to search the gene ontology database to collect the relevant biological processes and the GO terms. A higher stringency was imposed by considering only the GO terms with p < 0.001 as significantly enriched. Where GO terms were not found for certain bovine miRNAs, closely related and annotated miRNAs in other species were used as functional homologs.

### RESULTS

### Identification of Candidate miRNAs and Gene Targets Associated With Bovine Trypanosomosis

Our data mining revealed a total of 25 genes which were significantly regulated during bovine trypanosomosis. From the prediction analysis, we identified 4,251 bovine miRNAs that target these 25 genes spanning the 30UTR, 50UTR and the CDS regions (**Table 1**). The number of miRNAs per gene ranges from 32 as seen in LY96 to 578 in MAPKAPK3 gene. As shown in **Figure 1** and **Table 1**, about 8 other genes showed a higher density with over 200 miRNAs. It is observed that ICAM-1 and ITGAM genes had similar miRNA number (220).

In order to understand the distribution of the miRNAs across the nucleotide sequence of each gene, we performed miRNAtarget prediction for the 30UTR, 50UTR and the CDS regions using miRWalk. Generally, the CDS region presents the highest number of miRNAs for most genes followed by the 30UTR, while the 50UTR has the lowest (**Figure 2**). We observed that genes like LY96 and ITGAM have no miRNA at both 30UTR and 50UTR; CD80, IL-4 and IL-18 have no miRNA at the 3 0UTR, with IL-12A, IFN-γ, IL-1- and MYD88 presenting no miRNA at the 50UTR.

To examine the most conserved miRNAs across the 2 or 3 regions, we compared the overlapping miRNAs for each gene, with the assumption that miRNA present at two or three regions will be conserved with important biological inferences. Notably, there are more matches of miRNAs between the 30UTR and CDS region with FcγR3A, CD14 and MAPKAPK3 genes presenting 18, 20 and 99 matches respectively while CD80, IL-4, IL-18, LY96, ITGAM and IL-12A had no match of miRNA at the 3 regions (**Table 2** and **Supplementary Table S2**). Overall, we

13http://geneontology.org/

observed that only CXCL-8, TLR-4, and MAPKAPK3 genes had conserved miRNAs present in their 30UTR, 50UTR and the CDS regions; bta-miR-2454-5p in CXCL-8, bta-miR-2328- 3p in TLR-4, bta-miR-1777a, bta-miR-652 and bta-miR-7863 in MAPKAPK3 (**Supplementary Table S2**).

### Conserved Bovine miRNAs With Different Chromosomes

We gathered miRNA from genes located on the same chromosome and we identified common miRNAs which may give biological relevance to our study. In all, we found a total of 15 out of 25 selected genes located on the same chromosome with chromosome 7 having 4 genes (CSF2, CD14, ICAM-1, and IL-4), followed by chromosome 1 possessing 3 genes (CD80, CD86, and IL-12A) while chromosomes 3, 5, 22 and 23 have 2 genes each in common (**Figures 3A–F**). MAPKAPK3 and MYD88 on chromosome 22 has the highest number of common miRNAs with a total of 130; while TNF-α and CD83 on chromosome 23 have 53 miRNAs in common. Thirty common miRNAs are presented between CD80, CD88, and IL-12A.

### Network Analysis of Bovine miRNA-Target Interactome During Bovine Trypanosomosis

miRNA-target network was constructed in order to identify significant association and connections between miRNAs and different targets during bovine trypanosomosis. Out of the 25 selected genes and 4,251 miRNAs that were subjected to network analysis, only 13 genes and 54 miRNAs were significant connected in our network (**Figure 4**). Overall, based on the size of each node, CD14, MYD88, TNF-α, and IL-10 were significant nodes detected from the network analysis. Likewise, bta-mir-2888, bta-mir-2394, bta-mir-1284, and btamir-2467-p were major miRNA nodes connecting two or more genes in the network.

### Prediction of Conserved miRNAs-Target Interaction in 7 Innate Immune Response Genes

We focused on identifying miRNAs that are concomitantly present in the 7 innate immune response genes among the list of 25 genes that are significantly responding to bovine trypanosomosis. Here, we postulate that bovine miRNAs targeting the greatest number of genes will be potential candidates as biomarkers for disease diagnosis or treatment. From **Tables 3**–**5**, we present the prediction analysis of miRNAtarget interactions and their matured sequences. We showed the top 10 bovine miRNAs and their targets based on the 30UTR, 5 0UTR, and the CDS regions. Our selection is based on the miRNAs that target between 3-5 genes out of the selected 6 innate immune response genes. We observe that the CDS region has miRNAs that target the most genes; 5 out of the 7 genes at a time. For example, bta-miR-2349 targets CD14, ITGAM, TLR-2, TLR-4, and TNF-α while bta-miR-193a-5p targets ICAM-1, ITGAM, LBP, TLR-2, and TNF-α (**Table 3**). Following the CDS region is the 30UTR, while the 50UTR has the least number of

14http://diana.imis.athena-innovation.gr/DianaTools/index.php

TABLE 1 | List of significantly regulated genes during bovine trypanosomiasis and the number of miRNA at the 30UTR, 50UTR and CDS regions.

Significantly regulated genes were arranged in order of chromosome number (lowest to the highest).

miRNAs, targeting less than 4 genes (**Tables 4**, **5**). bta-miR-2460 from the 30UTR is demonstrated to target CD14, ICAM-1, TLR-4, and TNF-α genes, while bta-miR-2374 are targeting CD14, ICAM-1, LBP, and TLR-4 gene sets (**Table 4**), indicating they are the topmost in this region. Only bta-miR-2392 is targeting ICAM-1, TLR-2, TLR-4 and TNF-α from the 50UTR (**Table 5**).

**Table 6** shows the number of miRNAs common to a gene pair from each region. Our analyses reveal that the highest number of bovine miRNAs common between two genes are located at the CDS region. There are 55 miRNAs shared between TLR-4 and ITGAM, 54 between LBP and ITGAM, 52 between TLR-2 and ITGAM, 44 between ITGAM and ICAM-1, and 40 between TNF-α and ITGAM. Notably, CD14 and TNF-α have the highest common miRNA (15) at the 30UTR followed by 10 between CD14 and TLR-4 (**Table 6**).

### Study of Evolutionarily Conserved Bovine miRNA Among the 7 Innate Immune Response Genes and Other Species

Phylogenetic analyses were performed to examine the evolutionarily conserved bovine miRNAs with other species. To be more stringent, only miRNAs that target 4 out of 7 innate immune response genes were included in the analysis (one from the 50UTR, 2 from 30UTR and 10 from the CDS). Analysis of all the 10 miRNAs at the CDS with their homologous sequences from other species identified bta-mir-193a as highly conserved with human, monkey, dog, pig, chicken, gorilla, mouse, rat, rabbit, and alligator (**Figure 5**). In the same manner, bovine bta-mir-4657 clusters with a human homolog hsa-mir-4657, bta-mir-2888-2 with hsa-mir-6803 and bta-mir-2349 with hsa-mir-4769 (**Figure 5**). Furthermore, we individually searched for the conserved homologs of the 13 miRNAs selected for stringency (one from the 50UTR, 2 from 30UTR, and 10 from the CDS) in order to determine their evolutionary trace with other species. **Figures 6A–M** show different clusters of each miRNA with their homologs from other species. Based on the number of homologs found with other species, bta-mir-2888-1 (**Figure 6J**) with 14 homologs has the highest followed by bta-mir-193a with 13 (**Figure 6A**), bta-mir-2888-2, bta-mir-2392, and bta-mir-2349 have 11, 9 and 8 homologs respectively (**Figures 6E,G,K**).

TABLE 2 | Overlapping bovine miRNAs at the 30UTR, 50UTR and CDS.

UTR, untranslated region; CDS, coding sequence; red, indicate genes with matches and the numbers at the 30UTR, 50UTR and CDS regions.

bta-mir-2422 presented the least homolog with rat (rno-mir-327) and mouse (mmu-mir-327) (**Figure 6H**).

### Enrichment Analysis and GO Terms of the Conserved miRNAs

In order to better elucidate the possible biological and physiological processes regulated by the identified bovine miRNAs, 19 predicted conserved miRNAs were subjected to GO term analysis using the sub-term biological process as the focus. Topmost GO terms were selected for each tested miRNA as significantly enriched based on the p-value of less than 0.001. From the analysis, we found only 10 out of the 19 bovine miRNAs being functionally annotated with GO terms (**Table 7**). We infer the functional annotation of the remaining 9 bovine miRNAs from closely related species (in parentheses) based on the homology between 95-100%. We found that btamir-2460, bta-mir-193a, bta-mir-2328-3p, bta-mir-652, bta-mir-2392, and bta-mir-2454 are significantly involved with gene silencing (GO:0035195) and RNA-induced silencing complex (GO:0016442); bta-mir-2295 is involved with signal transduction in response to DNA damage (GO:0042770); bta-mir-2422 is involved with regulation of humoral immune response mediated by circulating immunoglobulin (GO:0002923), and bta-mir-2888-2 is involved with 2-aminobenzenesulfonate metabolic process (GO:0018868), among others (**Table 7**).

### DISCUSSION

Identification of molecular biomarkers associated with diseases is important in early disease diagnosis, drug target and treatment. miRNAs are increasingly emerging as molecular biomarkers because of their essential role in gene regulation both in animals and plants during disease condition (Buza et al., 2014; Agarwal et al., 2015; Scheel et al., 2017). In this study, we identified and elucidated conserved miRNAs from a list of gene targets important in innate immune response during bovine trypanosomosis through computational prediction analysis. Previous studies have focused on the 30UTR region to study the miRNA-target interactions while few others suggested that miRNAs can anneal within the 30UTR, 50UTR or CDS regions of their target to regulate gene expression (Erhard et al., 2014; Yang et al., 2017; da Silveira et al., 2018). This study however brings a unique perspective through identification of conserved miRNAs targeting immune genes responding during bovine trypanosomosis by analyzing the complete sequence of the gene which include the 30 -, 50 UTRs and the CDS. Our study showed a significantly higher density of miRNA binding sites at the CDS region for all genes, identifying it as an important region which may play a significant regulatory role in expression during the disease condition. Studies have shown

that target genes having many miRNA binding sites at the CDS region can be easily degraded (Schnall-Levin et al., 2011; Hausser and Zavolan, 2014). Likewise, Ott et al. (2011) and Chi et al. (2009) discovered that there are increasing miRNA target sites at the CDS region of mammalian transcripts during immunoprecipitation studies. This observation is in line with our results, suggesting that CDS sites may have specific function in regulating gene expression rather than the previous focus on the 30UTR (Hausser et al., 2013). Brummer and Hausser (2014) stated that the CDS sites may enhance the repertoire of miRNA regulation than the 30UTR sites because of their interaction with alternative polyadenylation, alternative splicing and the general gene architecture.

In addition, our study identified significant overlaps of miRNA binding sites between the 30UTR and CDS regions, indicating possible evolutionary conservation of these regulatory elements between the two regions (Hurst, 2006; Gaidatzis et al., 2007; Forman et al., 2008). Published reports have shown that miRNAs simultaneously targeting both 30UTR and CDS regions of mRNAs are more regulated than those targeted by 3 0UTR only (Fang and Rajewsky, 2011; Bazzini et al., 2012). Therefore, we adjudge that conserved miRNAs found targeting both regions have undergone selective pressure, and are therefore evolutionarily conserved, which may induce mRNA translational inhibition and degradation of immune gene response during bovine trypanosomosis.

FIGURE 5 | Phylogenetic tree of 13 bovine miRNA and their homologs in other species. The evolutionary trace was inferred using the Neighbor-Joining method. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the p-distance method and are in the units of the number of nucleotide sequence differences per site. The analysis involved 77 nucleotide sequences.

Furthermore, we found significant miRNAs cluster for genes located on the same chromosome, the most indication suggesting evolutionary conservation and functional roles on their targets. Thus, there is the possibility that expression of two or more genes on the same chromosome can be co-regulated by a single miRNA, and this observation might be important for quantifying gene regulation during disease condition or as targets for treatment. Pande et al. (2018) and Hanif et al. (2018) support the finding that miRNAs clusters across cattle genome have variable functions through large evolutionary distance.

Additionally, our study showed networks between miRNAs and target genes, which are significantly connected during bovine trypanosomosis. From our network interactome, CD14, TLR-2, TLR-4, IL10 MYD88, TNF-α, bta-mir-2360, bta-mir-1835, bta-mir-2888, bta-mir-146b and bta-mir-2320-5p amongst others are important drivers and connectors within the network, indicating they are major players during disease condition. Studies have shown that molecular interactions between genes, proteins, RNA and other biological molecules contribute to cellular performance and homeostasis (Govindaraju et al., 2012; Ji et al., 2018; Wang et al., 2018). The co-expression of these miRNAs within the network may regulate the interaction of certain gene sets such as MYD88 and TNF-α, CD14 and TLR-2, IL-18, and TLR-4, among others. Studies to date have demonstrated the importance of CD14 and TLR gene family in facilitating pattern recognition of pathogen-associated molecules and to elicit innate immune response against viruses, bacteria, fungi, and protozoa (Seabury et al., 2010; Ojurongbe et al., 2019; Morenikeji and Thomas, 2019b). Likewise, MYD88 is a signal transduction adaptor, transferring signals from interleukin-1 (IL-1) receptors and Toll-like receptors, and driving macrophage release of cytokines, imperative in innate immune response to exogenous pathogenic stimuli. Therefore, a significant overlap of miRNAs targeting these genes altogether could work synergistically as putative markers for disease discovery, characterization and therapeutic manipulations for bovine trypanosomiasis treatment.

Moreover, among the 7 innate immune response genes considered in this study, our functional analysis show that bta-mir-2460, bta-mir-193a, bta-mir-2349, bta-mir-3602, btamir-2454, and bta-mir-652 are significantly enriched within biological processes such as gene silencing, RNA-induced silencing complex, and cellular protein modification process. These miRNAs can potentially regulate CD14, ITGAM, LBP, TLR-2, TLR-4, and TNF-α genes and their expression might be key regulatory factors that determine disease susceptibility, tolerance or resistance in cattle. We have shown that CD14 is particularly implicated in disease tolerance among cattle with bovine trypanosomosis, with significant expression

bta-mir-3602; M: bta-mir-4657).

among trypanotolerant animals and the reverse among trypanosusceptible ones (Morenikeji and Thomas, 2019a). Notably, some studies have also shown that bta-mir-2460, bta-mir-193a, and bta-mir-652 are significantly involved in biological regulation, cellular and metabolic process, cell death, establishment of localization, and growth, thereby supporting their significant role in disease development (Jin et al., 2013; Schanzenbach et al., 2017; Li et al., 2018).

The significant number of targets by each miRNA depicts evidence of their evolutionary conservation with the implication that they play an essential role in gene regulation during bovine trypanosomosis. Yang et al. (2012) and Zheng et al. (2014) have reported the significant role of miRNAs in finetuning innate immune gene expression in Holstein cows during heat stress. Remarkably, bta-mir-2888-1 and btamir-2888-2 together targets ICAM-1, ITGAM, LBP, TLR-2, and TNF-α to respond to dihydrosphingosine-1-P pathway and 2-aminobenzenesulfonate metabolic process, which are important regulators of physiological biosynthesis, cell survival, proliferation, and cell-cell interactions (Bu et al., 2008). Previous studies have shown that ICAM-1, TLR-4 and ITGAM were implicated in early inflammation and signaling in critical pathways regulating bovine innate immunity (Boulougouris et al., 2019). As seen from our study therefore, bta-mir-2888-1 and bta-mir-2888 would be significant biomarkers that regulate the expression of these genes during disease condition and could serve as potential therapeutic targets for bovine trypanosomosis.

Our phylogenetic analysis revealed bta-mir-193a to be highly conserved across different species ranging from human to gorilla, monkey, mouse, rat, rabbit, chicken, pig, dog, python, and zebra. This shows that bta-mir-193a has a long history of evolution among others and would be a molecular marker of choice in bovine disease studies, and as such require additional in vitro validation studies. Reports from other studies have shown that bta-miR-193a regulates the expression of many proinflammatory cytokines, promote apoptosis and inhibition of bacteria in mice, goat and cattle (Dilda et al., 2011; Rosenberger et al., 2012; Lawless et al., 2013; Li et al., 2018). The fact that our results show bta-mir-193a targeting CD14, ITGAM, TLR-2, TLR-4, and TNF-α strongly supports its roles in transcriptional activation of proinflammatory response and innate immune gene expression during bovine trypanosomosis. This provides compelling evidence that this miRNA could serve as a significant biomarker in disease diagnosis and treatment. Many in vitro studies have elucidated the significant roles of miRNAs in regulating bovine immune responses during disease conditions (Dilda et al., 2011; Lawless et al., 2013; Wang et al., 2017; Ammah et al., 2018). The fact that TLR-2 and TLR-4 are part of btamiR-193a target suggests its involvement in Toll-like receptor signaling during host response to trypanosomosis infection.

### CONCLUSION

Our study provides computational evidence that certain miRNAs are conserved within the bovine genome and may be

### REFERENCES

associated with regulation of immune response during bovine trypanosomosis. We propose that miRNAs targeting more genes will play greater roles in immune regulation and as such exert significant impact on disease phenotype. We also show that the CDS region has abundant miRNA binding site for all genes in this study demonstrating the importance of this region and requiring further elucidation in bovine disease studies. Additionally, we demonstrate that miRNA that co-target 30UTR and CDS are important and capable of impacting target gene expressions in a complementary manner. Finally, our study identified some microRNAs including bta-mir-193a, bta-mir-2460, bta-mir-2349, and bta-mir-2888 amongst others, which might be important regulatory markers for diagnosis of bovine trypanosomosis as well as treatment and drug targets.

### DATA AVAILABILITY

All datasets generated for this study are included in the manuscript and/or the **Supplementary Files**.

### AUTHOR CONTRIBUTIONS

OM and MH performed the experiment. OM, MH, AH, and BT analyzed the data. OM, AH, and BT wrote the manuscript. All authors reviewed and approved the final manuscript.

### FUNDING

We acknowledge the intramural funding through a Laboratory and Faculty Development Award, College of Health Sciences and Technology, Rochester Institute of Technology (BT). OM is supported through the American Association of Immunologists Careers in Immunology Fellowship Program. AH acknowledge the College of Science and The Thomas H. Gosnell School of Life Sciences for ongoing support. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2019.02010/full#supplementary-material

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Morenikeji, Hawkes, Hudson and Thomas. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# *Trypanosoma brucei* Secreted Aromatic Ketoacids Activate the Nrf2/HO-1 Pathway and Suppress Pro-inflammatory Responses in Primary Murine Glia and Macrophages

#### *Edited by:*

Jude Ezeh Uzonna, University of Manitoba, Canada

#### *Reviewed by:*

Meiqing Shi, University of Maryland, College Park, United States Philippe Vincendeau, Université de Bordeaux, France Stefan Magez, Vrije University Brussel, Belgium

> *\*Correspondence:* Aisling Dunne<EMAIL_ADDRESS>
†These authors have contributed equally to this work ‡Co-authorship

#### *Specialty section:*

This article was submitted to Microbial Immunology, a section of the journal Frontiers in Immunology

*Received:* 30 May 2019 *Accepted:* 27 August 2019 *Published:* 11 September 2019

#### *Citation:*

Campbell NK, Williams DG, Fitzgerald HK, Barry PJ, Cunningham CC, Nolan DP and Dunne A (2019) Trypanosoma brucei Secreted Aromatic Ketoacids Activate the Nrf2/HO-1 Pathway and Suppress Pro-inflammatory Responses in Primary Murine Glia and Macrophages. Front. Immunol. 10:2137. doi: 10.3389/fimmu.2019.02137 Nicole K. Campbell 1†, David G. Williams 1†, Hannah K. Fitzgerald1 , Paul J. Barry 1 , Clare C. Cunningham1 , Derek P. Nolan1‡ and Aisling Dunne1,2 \* ‡

1 School of Biochemistry and Immunology, Trinity College Dublin, University of Dublin, Dublin, Ireland, 2 School of Medicine, Trinity Biomedical Biosciences Institute, Trinity College Dublin, Dublin, Ireland

African trypanosomes, such as Trypanosoma brucei (T. brucei), are protozoan parasites of the mammalian vasculature and central nervous system that are best known for causing fatal human sleeping sickness. As exclusively extracellular parasites, trypanosomes are subject to constant challenge from host immune defenses but they have developed very effective strategies to evade and modulate these responses to maintain an infection while simultaneously prolonging host survival. Here we investigate host parasite interactions, especially within the CNS context, which are not well-understood. We demonstrate that T. brucei strongly upregulates the stress response protein, Heme Oxygenase 1 (HO-1), in primary murine glia and macrophages in vitro. Furthermore, using a novel AHADHin T. brucei cell line, we demonstrate that specific aromatic ketoacids secreted by bloodstream forms of T. brucei are potent drivers of HO-1 expression and are capable of inhibiting pro-IL1β induction in both glia and macrophages. Additionally, we found that these ketoacids significantly reduced IL-6 and TNFα production by glia, but not macrophages. Finally, we present data to support Nrf2 activation as the mechanism of action by which these ketoacids upregulate HO-1 expression and mediate their anti-inflammatory activity. This study therefore reports a novel immune evasion mechanism, whereby T. brucei secretes amino-acid derived metabolites for the purpose of suppressing both the host CNS and peripheral immune response, potentially via induction of the Nrf2/HO-1 pathway.

Keywords: trypanosomes, keto acids, immune suppression, macrophages, glia

## INTRODUCTION