**3. Role of cytokines (TGF**β **and IL-6) in regulation of T-reg/Th-17 balance in malaria**

TGFβ and IL-6 are cytokines that play major roles in the regulation of innate and adaptive immune responses in different viral (viz. influenza A, Respiratory Syncytical virus etc.), bacterial (viz. *Streptococcus, Mycobacterium* etc), parasitic (viz. *Leishmania*, *Trypanosoma*, *Toxoplasma* etc.) infections, cancers and autoimmune disorders [44–47]. In malaria, IL-6 is found in circulation of patients infected with *Plasmodium vivax* and *Plasmodium falciparum* and it plays a major role in host response [48–50]. There are reports stating that decreased IL-6 levels upon treatment with anti-malarial compounds is associated with decreased parasitaemia [51–53]. However, several reports raise question on actual involvement of IL-6 in the pathogenesis of cerebral malaria [54–56]. In case of TGFβ, we have found that low concentration of TGFβ was found to be pro-inflammatory where high concentration of TGFβ have anti-inflammatory effects [41]. So, as factors responsible for disease outcome in malaria, both of these cytokines and their regulatory effect on T-reg/Th-17 balance seem to be important. We neutralized TGFβ and IL-6 by administration of neutralizing antibodies *in-vivo* at specific concentration. Parasitaemia was highest in TGFβ neutralized group than any other groups whereas parasitaemia was lowest in IL-6 neutralized group. This has been supported by the results of survival percentages of mice, where TGFβ neutralized group showed lowest survival percentage and IL-6 neutralized group showed the highest survival percentage of mice. Thus, it is quite evident that TGFβ and IL-6 directly affects the outcome of the immune response elicited by the host in malaria. Focusing on the effect of these two cytokines on the T-reg/Th-17 balance, it is found that neutralization of TGFβ results in significant induction of Th-17 cells at 8 dpi than control and infected ones. Whereas neutralization of IL-6 causes reduction in percentage and number of Th-17 cells than *Plasmodium berghei* ANKA infected group. Analysis of percentage and number of T regulatory cells in spleen show the reverse phenomenon to that of Th-17 cells upon neutralization of TGFβ and IL-6. Thus T-reg/Th-17 balance, which is skewed towards Th-17 in *Plasmodium berghei* ANKA infection is dependent on fine tuning maintained by TGFβ and IL-6. IL-6 neutralization reverts the dysregulated T-reg/Th-17 balance to homeostatic levels by inhibiting Th17 induction, but neutralization of TGFβ has opposing effect and causes the balance to skew more towards Th17. These changes in T-reg/Th17 balance by regulatory effects of TGFβ and IL-6 is mainly maintained by expression of STAT3 and STAT5, which are the major signalling molecules that take part in the signalling mechanism of these two cytokines [57]. Neutralization of TGFβ and IL-6 not only have its impact on splenic T-reg/Th-17, but also in that of cerebral cortex and cerebellum. In Anti-IL-6 treated *Plasmodium berghei* ANKA infected mice, glial cell mediated neuroinflammation is reduced whereas the anti-TGFβ treated mice upon infection show similar level of neuroinflammation as that of only infected mice. Consistent to that, astrocyte and microglia activation levels show similar changes in IL-6 and TGFβ neutralized groups. Regarding T-reg/Th-17, the major transcription factor of T-reg cells, FOXP3 expression was significantly higher in Anti-IL-6 treated infected group and significantly lower in Anti-TGFβ treated infected mice. The expression of IL-17, a major cytokine secreted by Th-17 cells, show the opposite result to that of FOXP3 in both the groups than the only

*Plasmodium berghei* ANKA infected ones [43]. But the actual percentages of the T-reg and Th-17 in cerebral cortex and cerebellum and their changes upon neutralization of these two cytokines is not yet investigated. Though there are few reports that cerebral malaria development is independent of IL-17 [58], several other reports shows that significant amount of IL-17 is found in circulation of malaria infected mice and human patients [59–61]. Genetic variants of IL-17 and its receptor IL-17RA increase the risk of malaria as investigated in African population [62]. Protective role of IL-17 during malaria pathogenesis has been found by working with IL-17RA deficient mice, in which IL-17 doesn't function in a proper way. These IL-17RA deficient mice show increased parasitemia, earlier onset of malaria, increased mortality during acute stage than the wild type mice [63]. So, it can be summarised that IL-17 itself and IL-17 expressing CD4+ T helper cells (Th17 cells) is of pivotal importance during malaria but the actual outcome of the immune response against the malaria parasite is dependent on the Treg/Th-17 balance, which is maintained majorly by TGFβ and IL-6.
