Dissertation: Impact of Vitamin D and RA on HLA-DR Levels in KG-1

This dissertation investigates the effects of Vitamin D and Retinoic Acid (RA) on HLA-DR levels using KG-1 cell lines. The study explores the interactions between vitamin D and RA, noting that their combined treatment decreased HLA-DR expression, contrasting with previous findings. The research also examines the role of CD38, an ectoenzyme, in the context of Vitamin D and RA treatments. The findings highlight the interconnected pathways of Vitamin D and RA, potentially influencing autoimmune disorders through HLA-DRB1*15 expression. The study compares its results with a similar project, revealing discrepancies and discussing the potential reasons behind these differences, including the amount of DMSO used and the overall effect of Vitamin D and RA. The conclusion emphasizes the regulatory role of Calcitrol and RA in immune functions, suggesting further research on the interaction between these pathways and the impact on autoimmune diseases.

Running head: DISSERTATION
DISSERTATION
Name of the Student:
Name of the University:
Author Note:

1DISSERTATION
Discussion:
The purpose of this chapter is to investigate the effect of the Vitamin D and RA on HLA- DR
levels in live ell lines. It should be noted in this context that this research is novel and forms
the first kind of research that evaluated this kind of interaction with any cell line. For this
purpose, KG- 1 cell lines were used for this investigation.
This impact of vitamin D which has on the HLA-DR is happens due to the interaction with
RA. On the other hand, MHC-II protein expression can be studied and evaluated by the
expression of the CD 38 cells. Therefore, the expressions of the CD 38 cells were studied in
this investigation so that the expression and effect of Vitamin D and RA can be studied.
The HLA-DR levelsresponded differentially to separate or combined treatment with vitamin
D and RA on the KG- 1 cell lines. The effect of 10nM vitamin D and 1uM retinoic acid on
HLA-DR levels can be observed in the Figure 3.6. From the graph it can be seen that the
percentage of HLA-DR expression levels was almost similar between vitamin D and DMSO.
The combined treatment of retinoic acid and vitamin D showed a decline in expression level
by 33 percent, while retinoic acid showed a decrease in expression levels by 34 percent. Two
way ANOVA suggests that this finding is significant with a p value less than 0.000. There is
no significant difference in case of the cells treated with ATRA. This is in clear contrast with
the study conducted by Shah(2018). In his study, in case of the myeloid cell line, Shah (2018)
observed that KG-1 combined treatment with vitamin D and RA led to the synergistic
lowering of the HLA-DR expression. As mentioned above, impact of vitamin D on HLA-DR
level occurs due to the interaction with RA (Villegas-Ospina et al. 2017). This mechanism
might be able to explain the above stated results. This also suggests that there is aninter-
connected relationship between the pathways of Vitamin D and RA and one pathway might
stop if another one is activated. Shah (2018) has also reported that there is an

2DISSERTATION
unresponsiveness of KG- 1 cells towards the dosage of biological addition of Vitamin D. they
have reported that there is no detectable effect Vitamin D on HLA- DR level. Similar like this
study, KG- 1 cells were also treated with the calcitriol and DMSO. This is in clear contrast of
the findings reported in this study and this study has reported a total opposite findings. Shah
(2018) has also stated in his study that HLA- DR expression reduced in case of KG- 1 cells
which were treated with Vitamin D and RA. Similar like the previous findings, this also in
contrast with the findings reported in this study. In this study, the expression levels increases
in addition with the biological Vitamin D. This difference in findings might be explained
through the amount of DMSO used in both of the studies. Amount of DMSO used is higher in
this study compared to study conducted by Shah (2018) and which might be reason behind
the changes in expression level. The overall effect Vitamin D and RA between the studies is
also different.
In addition to this, research studies have reported the presence of functional VRDE within the
promoter region of HLADRB1*15. The expression of HLADRB1*15 greatly increased upon
the addition of biologically active vitamin D. This critically suggests that a functional link
exists between the environmental and genetic factor that synthesises Vitamin D (Wergeland
et al. 2016). Therefore, it can further be concluded that lower expression of HLADRB1*15
would be triggered by the deficiency of vitamin D and this would increase the risk of
developing autoimmune disorders on account of the deletion of auto reactive T cells.
According to the Ramagopalan et al. (2010) Vitamin D deficiency leads to the limited
expression of HLADRB1*15. As a direct consequence of this mechanism, it leads to the
lowered deletion of the T-cells which will only increase the chance of autoimmune diseases.
Also, KG-1 cells have active turnover of the HLA-DR molecules which suggests that post
translational effect of RA and vitamin D could be potentially observed in these cells (Smith et
al. 2018). Various studies have reported the fact that HLA-DR expression enhances on the

3DISSERTATION
addition of biological Vitamin D, albeit, there is no consistent proof of this (Lang and
Aspinall 2017). Hence, it can be stated that the regulation of the allele DRB1*15 plays a
significant role for the regulation of the Vitamin D and therefore, Vitamin D might be able to
play a significant role as a biological protector in body (Simpson, der Mei and Taylor 2018).
On the other hand, HLADRB1*15 is plays a hand on the autoimmune disease like multiple
sclerosis (Rhead et al. 2016). The allele HLADRB1*15 has been associated with the
incidence of multiple sclerosis, however, HLADRB1*15 is not the only allele which is
responsible for this disease and alleles like DRB1*04:05 and DRB1*16:0 also plays a role in
this case (Cocco et al. 2012). Similarly, studies have reported that treatment of Vitamin D has
an effect on the HLA- DR levels on Type 1 diabetes patients as well (Carvalho et al. 2015).
The reason behind this mechanism is same as the case with the multiple sclerosis mentioned
above.
CD 38 can be defined as an ectoenzyme which generates during the cyclic ADP (adenosine
di- phosphate) ribose. CD 38 cells generally express on both T cells and B cells. However,
the exact role and function of the CD 38 cells in human are unknown still today (Deshpande
et al. 2017). Therefore, there is a need for the further study in this area to properly investigate
the function of CD 38 cells which might shed some light for better understanding of the
pathophysiology of auto immune diseases and its treatment (Wang et al. 2014). In this
investigation, it has been found out that the CD 38 cells were induced at very low level and
they were only induced by the ATRA and vitamin D had no effect at all on the induction of
the CD 38 cells. Studies have reported that ATRA generates robust expression on the CD 38
cells (Wang et al. 2014). Forced myeloid differentiation might be the reason behind this and
it can also be implied to this study which also investigated with the myeloid cell line KG- 1
(Wang et al. 2014).

4DISSERTATION
In support of the finding, a research study has reported thatan increase of the
DRB1*15 gene transcription of the PGF cells showed an increase in level of the protein
expression after a treatment with vitamin D (Ramagopalan et al. 2009). The similar findings
were detected in this research study as well with increase in expression level for Vitamin D
and DMSO. Overall approximately, 20 to 25% of an increase in the HLA-DR expression was
observed and detected in the PGF and WDV cells (Backdash et al. 2015). In this study, the
increment in expression was at around 8 percent. Further, the PGF cells responsible for the
expression of HLA-DRB1*15 expressed an alternate beta chain gene, DRB5 at a higher level
(Wesolowskietal. 2013). On the other hand, DRB1*15 gene product is expressed at reduced
levels at the mRNA and the protein level (Roche et al. 2015). According to Ramagopalan et
al. (2009), vitamin C acts on the VDRE positioned at the HLA-DRB1*1201 promoter region
to enhance its expression. This has ultimately been reported to increase the total HLA-DR
expression by approximately 50% within the PGF cells (Roche et al. 2015). In comparison to
this study, cells stained with fluorescent antibodies IgG2a-FITC have higher expression
levels of antigens by 67%. The expression of DRB1 has been reported to increase but the
expression of DRB5 has been reported to be constant. It can therefore be expected that the
difference in the expression level of HLA-DR on account of treatment with vitamin D helped
in forming an understanding about a number of genetic variants in DRB1*15:01
(Hauser,Oksenberg and Baranzini 2015). Typically a significant increase was observed in the
expression level of HLA-DR antigens for the cells which were stained with IgG2a-FITC (67
percent increase). In addition to this upon the combined use of Vitamin D and DMSO, an 8
percent more increment in the expression level of HLA-DR antigen was noted whereas
Retinoic acid causes a decrease in HLA-DR expression levels in KG-1 cells. Overall,
Vitamin D does not appear to influence the antigen expression levels.
Conclusion:

5DISSERTATION
Therefore, to conclude it can be mentioned that Calcitrol regulates a wide range of
immune functions such as regulation of shift from the pro- inflammatory to a tolerogenic
immune status. Retinoic Acid on the other hand, is responsible for the expression of a number
of genes which are tightly linked to the immune cells. Retinoic acid further plays an
important role in modulating the mechanism of switch between the regulatory as well as the
inflammatory T-cells. The HLA class II molecules that are synthesized by the rough
endoplasmic reticulum are present within the B cells, macrophages as well as the dendritic
cells. Through the study, the effect of vitamin D on the expression level of HLA-DR was
evaluated. However, it should be noted that level of interaction of the HLA-DR with the
Retinoic Acid regulated the level of HLA-DR expression.The effect on the level of
expression of HLA-DR upon the addition of RA and vitamin D was increased as was evident
in KG- 1 cell lines. This clearly suggests that a mutual interaction exists between the two
pathways where activation of one pathway stops the second pathway from increasing the
level of expression. The limitation of this study is that this study cannot shed any definitive
light on the type of interaction between them. This further serves as an interesting area of
future research for researchers. Furthermore, the study does not shed light on the possibility if
more than one copy number variant is present and this also needs further research. Future
research should be conducted to study the type of interaction exists between expression of
HLA-DR upon the addition of RA and vitamin D.

6DISSERTATION
References:
Bakdash, G., Vogelpoel, L.T., Van Capel, T.M., Kapsenberg, M.L. and de Jong, E.C., 2015.
Retinoic acid primes human dendritic cells to induce gut-homing, IL-10-producing regulatory
T cells. Mucosal immunology, 8(2), p.265.
Carvalho, C., Marinho, A., Leal, B., Bettencourt, A., Boleixa, D., Almeida, I., Farinha, F.,
Costa, P.P., Vasconcelos, C. and Silva, B.M., 2015. Association between vitamin D receptor
(VDR) gene polymorphisms and systemic lupus erythematosus in Portuguese
patients. Lupus, 24(8), pp.846-853.
Cocco, E., Meloni, A., Murru, M.R., Corongiu, D. and Tranquilli, S., 2012. Vitamin D
Responsive Elements within the HLA-DRB1 Promoter Region in Sardinian.
Deshpande, D.A., Guedes, A.G., Lund, F.E., Subramanian, S., Walseth, T.F. and Kannan,
M.S., 2017. CD38 in the pathogenesis of allergic airway disease: Potential therapeutic
targets. Pharmacology & therapeutics, 172, pp.116-126.
Hauser, S.L., Oksenberg, J.R. and Baranzini, S.E., 2015. Multiple sclerosis. In Rosenberg's
Molecular and Genetic Basis of Neurological and Psychiatric Disease (pp. 1001-1014).
Academic Press.
Lang, P.O. and Aspinall, R., 2017. Vitamin D status and the host resistance to infections:
what it is currently (not) understood. Clinical therapeutics, 39(5), pp.930-945.
Ramagopalan, S.V., Heger, A., Berlanga, A.J., Maugeri, N.J., Lincoln, M.R., Burrell, A.,
Handunnetthi, L., Handel, A.E., Disanto, G., Orton, S.M. and Watson, C.T., 2010. A ChIP-
seq defined genome-wide map of vitamin D receptor binding: associations with disease and
evolution. Genome research, 20(10), pp.1352-1360.

7DISSERTATION
Rhead, B., Bäärnhielm, M., Gianfrancesco, M., Mok, A., Shao, X., Quach, H., Shen, L.,
Schaefer, C., Link, J., Gyllenberg, A. and Hedström, A.K., 2016. Mendelian randomization
shows a causal effect of low vitamin D on multiple sclerosis risk. Neurology Genetics, 2(5),
p.e97.
Roche, P.A. and Furuta, K., 2015. The ins and outs of MHC class II-mediated antigen
processing and presentation. Nature Reviews Immunology, 15(4), p.203.
Shah, N. 2018. Regulation of MHC II protein expression by lysosomal degradation and
Vitamin D. Ph.D. Roehampton University.
Simpson, J., der Mei, I.V. and Taylor, B., 2018. The Role of Vitamin D in Multiple Sclerosis:
Biology and Biochemistry, Epidemiology and Potential Roles in Treatment. Medicinal
Chemistry, 14(2), pp.129-143.
Smith, T.J., Wilson, M., Karl, J.P., Smith, C., Cooper, A., Heaton, K., Orr, J., Hruby, A.,
Young, A.J. and Montain, S.J., 2018. Impact of Sleep Restriction on Local Immune Response,
Skin Barrier Restoration, Cognition, Marksmanship and Gut Function with and
without'Multi-nutrient'Nutrition Intervention (No. 13-10H). US Army Research Institute of
Environmental Medicine Natick.
Villegas-Ospina, S., Aguilar-Jimenez, W., Gonzalez, S.M. and Rugeles, M.T., 2017. Vitamin
D modulates the expression of HLA-DR and CD38 after in vitro activation of T-
cells. Hormone molecular biology and clinical investigation, 29(3), pp.93-103.
Wang, Z., Liu, Z., Wu, X., Chu, S., Wang, J., Yuan, H., Roth, M., Yuan, Y.C., Bhatia, R. and
Chen, W., 2014. ATRA-induced cellular differentiation and CD38 expression inhibits
acquisition of BCR-ABL mutations for CML acquired resistance. PLoS genetics, 10(6),
p.e1004414.

8DISSERTATION
Wergeland, S., Myhr, K.M., Løken‐Amsrud, K.I., Beiske, A.G., Bjerve, K.S., Hovdal, H.,
Midgard, R., Kvistad, S.S., Holmøy, T., Riise, T. and Torkildsen, Ø., 2016. Vitamin D, HLA‐
DRB 1 and Epstein–Barr virus antibody levels in a prospective cohort of multiple sclerosis
patients. European journal of neurology, 23(6), pp.1064-1070.
Wesolowski, R., Markowitz, J. and Carson, W.E., 2013. Myeloid derived suppressor cells–a
new therapeutic target in the treatment of cancer. Journal for immunotherapy of cancer, 1(1),
p.10.