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\def\RunningAuthor{Fadlul Azim Fauzi Mansur et al.}
\firstPage{1539}
\articleType{Original Article}
\receivedDate{26 Aug 2020}
\acceptedDate{03 Oct 2020}
\revisedDate{26 Sep 2020}
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\journalDoi{ijrps.v11iSPL4.4335}
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\title{\textbf{The Relative Indirect Anthelmintic Effect of Caprine Milk on Mucins Gene Expression \textit{in Vitro} Using IL-22 Treated LS174T Cells Model of Helminth Infection}}
\author{Fadlul~A~F~Mansur\textsuperscript{*}\textsuperscript{1},  
            Anis~NA~Rauf\textsuperscript{1},  
            Nur~FM~Manzor\textsuperscript{1},  
            Faizul~H~Addnan\textsuperscript{1},  
            Wan~O~Abdullah\textsuperscript{1}, 
            Omaima~A~Najm\textsuperscript{2}~\\[5pt]{\textsuperscript{1}Faculty of Medicine and Health Sciences, Universiti Sains Islam Malaysia, Persiaran Ilmu, Putra Nilai 71800, Negeri Sembilan, Malaysia}~\\{\textsuperscript{2}Department of Biology, Colloge of Education for Pure Science, University of Mosul, Mosul, Iraq}}

\begin{abstract}
Mucus secretion by intestinal goblet cells constitutes an important mechanism in TH2 response following helminth infection mediated by the key cytokine IL-22. This indirect mechanism rather than directly attacking the parasite is important in preventing helminth attachment hence promoting helminth expulsion from the intestinal tract. We hypothesized that natural products having an anthelmintic activity like caprine milk may exert similar response. Using human intestinal LS174T cells treated with IL-22 to simulate helminth infection, we tested whether or not the co-treatment with caprine milk induces MUC1, MUC3, MUC4 and MUC5B genes expression. Optimal concentrations for caprine milk was determined to be 25\% and 50\% from cell viability assay. IL-22 induced helminth infection model was confirmed. However, the indirect anthelmintic effect of caprine milk was only relative as treatment of caprine milk in LS174T cells and IL-22 \textit{in vitro} did not significantly induce MUC1, MUC3, MUC4 and MUC5B genes expression when compared to treatment with IL-22 alone. In conclusion,\textbf{\space }caprine milk was not significantly associated with the mechanism of increased mucus production through upregulation of mucin genes by intestinal cells. Caprine milk may possess direct anthelmintic effect rather than indirect.
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}anthelmintic,\newline caprine milk,\newline in vitro,\newline IL-22,\newline LS174T
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Fadlul~A~F~Mansur~\\ Phone:\ +6-03-42892400~\\  Email:\ fadlul@usim.edu.my
        \par}\vspace*{-11pt}\hrulefill\par{\fontsize{12}{14}\selectfont ISSN: 0975-7538}\par% 
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\section{Introduction}
Intestinal parasitic helminth infection is a global affliction with significant disease burden \unskip~\citep{943799:20857795}. In order to survive within the human host, parasitic helminths have evolved various strategies to evade immune attack ranging from passive tactic like using blood group antigens as cover \unskip~\citep{943799:20857787} to actively modulating the immune system\unskip~\citep{943799:20857788}. The body's humoral response to such invasion has been the T-helper type 2 (TH2) response which involves mast cells and various cytokines. With regards to the intestine, such a response involves secretion of mucus to facilitate worm expulsion of weak or dead worms\unskip~\citep{943799:20857791}. Specific to hyperproduction of mucus are the role of goblet cells and upregulation of mucin genes by IL-13\unskip~\citep{943799:20857792} and IL-4 \unskip~\citep{943799:20857800}. Interestingly, Turner, Stockinger and Helmby in 2013 had demonstrated the key role of IL-22 in anthelmintic immunity, particularly in goblet cell function leading to worm expulsion which can be seen as an indirect anthelmintic activity compared to mostly direct effects\unskip~\citep{943799:20857803} mounted against worms in the intestine. The control of parasitic helminth infections is principally through chemotherapy \unskip~\citep{943799:20857794} but is hampered by the problem of resistance which led many to screen plant based products for anthelmintic activity\unskip~\citep{943799:20857783}. 

Animals are also potential sources for medicine. Porcine based therapies like clexane and insulin are hospital mainstays\unskip~\citep{943799:20857790}. Thrombolytic agents used in stroke like Ancrod was developed from snake venom\unskip~\citep{943799:20857786}. Experimentation on the anthelmintic effects of milk began during the post war era but quickly loses popularity shortly thereafter\unskip~\citep{943799:20857782}. Bovine milk exerted an anthelmintic effect causing a reduction in parasites when pigs were given skim milk\unskip~\citep{943799:20857806,943799:20857797,943799:20857796}. 

Moreover, bovine milk and milk products appear to be active \textit{in vitro} by reducing the motility of both sheathed and exsheathed L3 \textit{Ostertagia} in whey protein\unskip~\citep{943799:20857798}. The superior anthelmintic activity of camel milk against \textit{Haemonchus contortus} in comparison to milk from cow, ewe and goat were reported \textit{in vitro}\unskip~\citep{943799:20857804} and \textit{in vivo}\unskip~\citep{943799:20857805} who suspected the role of lactoferrin and vitamin C in mediating the effects. Caprine milk (goat milk), a popular drink in many parts of the world and many cases often being promoted as a functional food with many nutraceutical effects \unskip~\citep{943799:20857789}. 

Evidence of its anthelmintic activity is very limited with \unskip~\citep{943799:20857804} documenting a statistically insignificant effect using \textit{in vitro} assay. The mechanism by which caprine milk exerts its anthelmintic activity is poorly understood with only \unskip~\citep{943799:20857784} observing a direct hydrolysing effect on worm cuticle from transmission electron micrographic study. Would caprine milk affect worms indirectly? We hypothesized that caprine milk may exert an indirect response towards helminth infection inducing the immune system to upregulate mucin genes. Using human  Dukes' type B colorectal adenocarcinoma cell line (LS174T) cells treated with IL-22 to simulate helminth infection, we tested whether or not the co-treatment with caprine milk induces mucin 1 (MUC1), mucin 3 (MUC3), mucin 4 (MUC4) and mucin 5B (MUC5B) genes expression by qPCR.
    
\section{Materials and Methods}
\textbf{Milk}

Frozen caprine milk was collected from Berkat Jaya farm in Sungai Buloh, Selangor, Malaysia. They were kept at -20\textbf{\ensuremath{^\circ}}C at Faculty of Medicine and Health Sciences, Universiti Sains Islam Malaysia (USIM) until use.

\textbf{Cell culture} \textbf{\space }

The human Dukes' type B colorectal adenocarcinoma (LS174T) (No. C0009013) was purchased from Addexbio (Addexbio, USA). Thawed cells in the vial were transferred into desired culture flask with complete culture Eagle's Minimal Essential Medium (MEM) (Gibco, USA)supplemented with 10\% fetal bovine serum (FBS) (Gibco, USA) and 1\% penicillin-streptomycin (Gibco, USA). For passaging adherent cells culture, the culture medium was discarded, and culture flask (Thermo Fisher, USA) was washed using 5 ml (for T75 culture flask) or 10 ml (for T125 culture flask) 1x Phosphate Buffer Saline (PBS) (Gibco, USA). The wash solution was then discarded before pre-warmed dissociation reagent, 0.25\% trypsin-EDTA (Gibco, USA) was added and incubated for 4 minutes in 37\ensuremath{^\circ}C incubators. 

Next, the flask was inspected and observed under a light inverted microscope (Olympus CKX 41, Germany) to ensure that cells have completely detached from the flask surface. The same amount of complete culture medium was added into the respective flask to deactivate the trypsin effect. The mixed media with cells was then transferred into 50ml microcentrifuge tube (Eppendorf, USA) and centrifuged at 1,500 x \textit{g } for 7 minutes. The cell pellet was resuspended in a minimal volume of pre-warmed complete culture medium and 90\ensuremath{\mu}l  was aliquoted for cell counting using trypan blue (Sigma-Aldrich, Germany), staining and haemocytometer (Hawksley, England). A total of 5 x 10\ensuremath{^{5}} cells would be an ideal amount for an overnight 80\% cell confluence in 6-well plate (Thermo Fisher, USA).


\bgroup
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\makeatother 
\caption{\boldmath {The effect of various concentrations of caprine milk on the rate of LS174T cell proliferation at 24 and 48 hours of incubation.}}
\label{f-cba180671a08}
\end{figure*}
\egroup

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\caption{\boldmath {MUC1(A\&B); MUC3(C\&D); MUC4(E\&F); MUC5B(G\&H) mRNA expression relative to the housekeeping gene, GAPDH, after 24 and 48 hours of treatment in different concentrations of caprine milk.}}
\label{f-14e71718b9fd}
\end{figure*}
\egroup

\begin{table*}[!htbp]
\caption{\boldmath {List of forward and reverse primers for mucin 1, mucin 3, mucin 4 and mucin 5B} }
\label{tw-df85855a804c}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.2502\linewidth-2\tabcolsep}p{\dimexpr.18339999999999996\linewidth-2\tabcolsep}p{\dimexpr.27689999999999998\linewidth-2\tabcolsep}p{\dimexpr.28950000000000003\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Gene name & Accession number & Forward primer & Reverse primer\\
\tblmidrule 
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) &
  99166649 &
  5'-ATCACCA \mbox{}\protect\newline TCTTCCAGGAGCGA-3' \mbox{}\protect\newline  &
  5'-AGCCTTCTCCAT GGTGGTGAA-3'\\
Mucin 1 (MUC1)  &
  99200183 &
  5'-ACAGTGCTTACA GTTGTTACGGGT-3' &
  5'-CCTGGCAGAGGT GCCGTTGT-3'\\
Mucin 3 (MUC3) &
  99200185 &
  5'-TGGATCTAGATG TAGTGGAGACC-3'  &
  5'-TGCAAAAATCCT CTGCATCTG-3'\\
Mucin 4 (MUC4) &
  99200187 &
  5'GCCCAAGCTAC \mbox{}\protect\newline AGTGTGACTCA-3'   &
  5'ATGGTGCCGT \mbox{}\protect\newline TGTAATTTGTTGT-3'\\
Mucin 5B (MUC5B)  &
  99200181 &
  5'GGGCCTCGAG \mbox{}\protect\newline TGCCGTG-3'  &
  5'CACACGGA \mbox{}\protect\newline TTCATAGTTGAA-3'\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}
\textbf{Cell viability assay}

The effects of caprine milk on LS174T cell proliferation or inhibition was analyzed using MTS assay kit (CellTiter 96{\textsuperscript{\textregistered}} Aqueous\ensuremath{_{}} One Solution Cell Proliferation Assay, Promega, USA). Approximately, 1x10\ensuremath{^{5}} cells/well were seeded in 96-well plates and allowed to attach overnight. Cells then were treated with different concentrations based on doubling dilutions (100\%, 50\%, 25\%, 12.5\%) of caprine milk and incubated at 37\ensuremath{^\circ}C in a humidified atmosphere with 5\% CO\ensuremath{_{2}} for up to 48 hours. 

Following the treatment period, 20\ensuremath{\mu}l of MTS reagent (Promega, USA) is added into 100\ensuremath{\mu}l of each well containing sample and incubated for 4 hours. Finally, absorbance of each well is recorded at 490nm using microplate reader (Tecan, Switzerland). Percentage of cell viability was calculated using the formula below;

\textbf{Cell culture model and treatment group}

The effects of caprine milk on mucin genes expression of LS174T cells were determined using IL-22-induced LS174T \textit{in vitro} model. The concentration of IL-22 for induction of helminth-like infection condition were optimised. The cell culture model were divided into 4 main groups which are treatment of cells with; (1) growth medium as control 1, (2) IL-22 alone as control 2, (3) caprine milk 50\% + IL-22 and (4) caprine milk 25\% + IL-22. Each treatment was performed for 24 and 48 hours duration. Upon completion of treatment, cells were harvested for RNA extraction and gene expression analysis.

\textbf{Quantitative real time PCR (qPCR)}

Cells were harvested and stored in TRIzol\textsuperscript{TM} (Invitrogen, USA)reagent at -80\ensuremath{^\circ}C until processing. RNA was purified using TRIzol\textsuperscript{TM}. cDNA was prepared via reverse transcription using SuperScript III First-Strand Synthesis Supermix for qRT-PCR (Invitrogen, USA) and quantitative PCR was performed using SSO Advanced Universal SYBR Green Supermix (Bio-Rad, USA). Protocols for cDNA synthesis and qPCR were run in Applied Biosystems\textsuperscript{TM} StepOne\textsuperscript{TM} Real-Time PCR System (Thermo Fisher Scientific, USA). All protocols; RNA extraction, cDNA synthesis and qPCR were based on manufacturers' recommendation. Results were normalised to the housekeeping gene GAPDH.

\textbf{Preparation of forward/reverse primers}

The primers for housekeeping gene, GAPDH, and gene of interest; MUC1, MUC3, MUC4 and MUC5B were synthesised based on sequences in Table~\ref{tw-df85855a804c}. The primers were reconstituted into 100\ensuremath{\mu}M primary stock using 1x TE buffer (pH 8) (Invitrogen, USA) and 100\ensuremath{\mu}M stock solution using Nuclease-Free Water (Thermo Fisher Scientific, USA).

\textbf{Data analysis}

Data were analyzed using Graphpad Prism 7 software (USA). Kruskal-Wallis test was used to test significant differences on the expressions of MUC1, MUC3, MUC4 and MUC5B between groups of LS174T cells receiving various concentrations of caprine milk and IL-22. A p-value of less than 0.05 was considered statistically significant.
    
\section{Results}
\textbf{Cell viability assay} \textbf{\space }

In this study, we tested few concentrations of caprine milk (100\%, 50\%, 25\%, 12.5\%) on LS174T cell viability using MTS assay. As shown in Figure~\ref{f-cba180671a08}, LS174T cells remain viable at 24 and 48 hours. From the viability test, 2 concentrations of caprine milk (50\%, 25\%) showed optimal cells viability hence chosen to proceed with the mRNA expressions of various mucin genes (MUC1, MUC3, MUC4, MUC5B) after treatment of LS174T cells with the cytokine IL-22.

\textbf{Mucins mRNA expressions after treatment of LS174T cells with caprine milk and IL-22} \textbf{\space }

Expressions of human mucins such as MUC1, MUC3, MUC4 and MUC5B were quantitated using real time qPCR. \ensuremath{\Delta }Ct was used to derived 2\ensuremath{^{-\ensuremath{\Delta }Ct}} based on protocol of Schmittgen \& Livak in 2008. There were no significant differences of MUC1, MUC3, MUC4 and MUC5B expressions between LS174T cells treated with IL-22 alone and cells treated with different concentrations of caprine milk (see Figure~\ref{f-14e71718b9fd}). 
    
\section{Discussion}
Our findings failed to observe a significant upregulation of mucin genes when intestinal cells (LS174T) were induced using IL-22 to simulate helminth infection \unskip~\citep{943799:20857802} and treated with caprine milk which is known to have anthelmintic activity \unskip~\citep{943799:20857804,943799:20857784,943799:20857789}. However, cells treated with IL-22 alone successfully upregulated MUC1 expression as compared to cells treated with media only (negative control) confirming the indirect anthelmintic expulsion model proposed by \unskip~\citep{943799:20857802} as well as strengthening the idea that IL-22 is the key cytokine in such a response. This is important as most work highlighted other TH2 \unskip~\citep{943799:20857793,943799:20857785}  cytokines such as IL-13 \unskip~\citep{943799:20857792} and IL-4 \unskip~\citep{943799:20857800} which modulates mast cells and macrophages \unskip~\citep{943799:20857801}. IL-22 was also evident in worm therapy experimentation using \textit{Trichuris trichiura} to treat ulcerative colitis patients \unskip~\citep{943799:20857799}.  

Our model (LS174T and IL-22) may be used by others who wish to mimic the indirect anthelmintic immune response of helminth infection modelled earlier by \unskip~\citep{943799:20857802} in which IL-22 alone was able to induce the expression of several goblet cell mediators hence increase mucin production by intestinal epithelium and assists in worm expulsion. However, in the present study we did not include positive control because there is no known substance has been shown to upregulate mucins gene expression. Although not statistically significant, our work demonstrated that caprine milk relatively induced mucin genes expression in a dose dependant manner for MUC1, MUC4 and MUC5B. Most mucins mRNA showed upregulation in at least one concentration of caprine milk treatment.

However, the upregulations were not statistically significant. Hence, there is no significant association of mucin expressions between LS174T cells treated with cytokine IL-22 (control) alone and cells treated with variations of caprine milk concentration. The variations between Ct values of the mucins obtained were quite high. This could contribute to the insignificant data results from this qPCR analysis. However, the trend of increased mucins gene expression from caprine milk treatment is noted and should be evaluated further. 
    
\section{Conclusion}
Human LS174T intestinal cells treated with IL-22 and caprine milk \textit{in vitro} did not induce MUC1, MUC3, MUC4 and MUC5B genes expression. Therefore, the anthelmintic effect of caprine milk is not associated with indirect effects of intestinal cells increasing mucus production through upregulation of mucin genes. Caprine milk may be exerting its anthelmintic effect directly onto the helminth cuticle proteolytically digesting them much like cysteine proteinases.
\section*{Acknowledgement}We thank Farhana Hamdan for technical help during the earlier stage of this study.

\textbf{Conflict of interest}

The authors declare that they have no conflict of interest for this study.

\textbf{Funding support}

This study was fully funded by Ministry of Higher Education of Malaysia under the Niche Research Grant Scheme [USIM/NRGS\_ P10/FPSK/ 8409/52113].
    

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