Shashi Baruah

Heightened inflammation in severe malaria is associated with decreased IL-10 expression levels and neutrophils

Dysregulation of the cytokine network in severe malaria owing to variations in factors like parasite load, strains and host factors is well documented but the key cytokines that are dysregulated remain poorly elucidated. Longitudinal changes in cytokine levels in an individual with parasitemia and disease resolution is likely to identify the key cytokines. We have analyzed the mRNA expression of cytokines over a 7-d period in severe (SM) and uncomplicated (UM) Plasmodium falciparum malaria. We found up-regulated expression of TNF-α, IL-1β, IFN-γ and TGF-β in SM, with decreased expression of IL-10 on d 0. Further, we observed a negative correlation of IL-10 expression with parasitemia and pro-inflammatory cytokines, suggesting IL-10 to be the key cytokine in tilting the balance to an inflammatory response. Longitudinal analysis revealed that the key cytokines associated with disease were TNF-α, IL-1β, IFN-γ, IL-12α, RANTES and TGF-β, while TNF-α, IL-10 and TGF-β discriminated between SM and UM. A higher neutrophil count in SM and its positive association with parasite density and IL-1β and IL-8 provides support for neutrophils in inflammation in malaria. Our findings suggest subversion of anti-inflammatory response in SM by parasite factors towards an exaggerated pro-inflammatory response with involvement of neutrophils, the classical inflammatory cells.

Temporal and spatial variation in MSP1 clonal composition of Plasmodium falciparum in districts of Assam, Northeast India

Polymorphism in MSP1 gene generated by insertion/deletion of repeats causing repeat length polymorphisms is widely used as a marker for parasite genotyping. Elucidating Plasmodium falciparum clonal composition in relation to transmission intensity and other epidemiological factors in endemic areas is crucial to understanding the dynamics of host-parasite relationship and the development of immunity in malaria. We have examined here the allelic diversity of P. falciparum and attempted to understand the polymorphism and distribution of alleles of MSP1 with transition in transmission season and with differences in malaria epidemiology between sites. MSP1 diversity expressed as mean number of distinct alleles per isolate was 0.68 at Dimakusi and was much higher (p=0.007) than seen at Guabari (0.336) and Kondoli (0.45) as was multiplicity of infection at 4.12, indicating the highest diversity at this site. Size polymorphism of the allelic families at Guabari was distinctly different from Kondoli but shared similarity with Dimakusi. Infections in high transmission summer season tended to be more complex with higher number of alleles. The frequency of alleles of RO33 and MAD20 allelic families at Guabari was found to be different between the two transmission periods. A 380 base pair allele of RO33 was over represented in high transmission summer season and seen frequently in isolates with high parasitaemia. At Kondoli allele distribution of only MAD20 was found to be different in each study year. Study site and ethnicity but not age of the study population were identified as risk factors in infection complexity. The present study demonstrates that allelic composition of P. falciparum varied with study site and between periods of high and low transmission as well as in different years of study.

Polymorphisms and expression of TLR4 and 9 in malaria in two ethnic groups of Assam, northeast India

Infectious diseases have been postulated to play an important role in exerting pressure and in selection of TLR polymorphisms. Single nucelotide polymorphisms (SNPs) of TLR4 have been reported to show unique distributions in populations from Africa, Asia and Europe, and malaria is suggested to influence these patterns. In this context, we examined association of TLR polymorphisms with the risk of malaria in two ethnic groups—the Austro-Asiatics and Tibeto-Burmans—from malaria endemic districts of Assam to understand the influence of malaria in selection of TLRs in these genetically-distinct populations. TLR9 (T-1237C) mutation was positively associated with complicated (P = 0.001) and frequent (P = 0.035) malaria in Austro-Asiatics (relative risk = 0.595 95% CI: 0.479–0.836), but not in Tibeto-Burmans. Nonetheless, these alleles were not in Hardy-Weinberg Equilibrium in Tibeto-Burmans (P < 0.001). In contrast, the TLR9 1486T/C genotype was favourable where it was negatively associated with complicated malaria (Fishers exact P = 0.014). Sequencing data revealed that the two populations differed in nucleotide diversity of the TLR9 promoter region. Enhanced expression of TLR4 (P = 0.05), but not of TLR9, was associated with complicated malaria. Austro-Asiatics appeared to have accumulated favourable genotypes of TLR9, perhaps because of their longer exposure to malaria.

Dysregulation of cytokines expression in complicated falciparum malaria with increased TGF-β and IFN-γ and decreased IL-2 and IL-12

BACKGROUND In Plasmodium falciparum infections, proinflammatory cytokine response is implicated in control of parasite multiplication as well as in disease pathogenesis. However, the regulation of proinflammatory and anti-inflammatory cytokine balance and its relation to disease severity remains poorly understood. METHODS We examined cytokines gene expression by quantitative real time-PCR technique in a case control study comprising of P. falciparum infected (n=58) and non infected (n=30) groups. P. falciparum infected were further stratified as complicated and uncomplicated as per WHO criterion and parasitaemia levels. RESULTS Higher expression of IL-2, IL-12α and TGF-β with decreased levels of IL-10 was seen in P. falciparum positivity. Complicated malaria was associated with enhanced expression of IFN-γ and TGF-β but lower IL-2 and IL-12α in comparison to uncomplicated malaria. Modeling of data suggested higher expression of IL-12α to be predictive of uncomplicated malaria [Odds ratio=3.074, 95% CI (1.254-7.536)] and was negatively associated with complicated malaria outcome (p=0.014). Interestingly, the probability of complicated malaria in males with elevated TNF-α expression was three times higher [p=0.05; Odds ratio=3.412, 95% CI (0.98-11.848)]. Age was also seen to be a factor with higher IL-8 in diseased young (p=0.012). CONCLUSION Our data suggested induction of balanced cytokine response in uncomplicated malaria while cytokine dysregulation with a role for TGF-β was indicated in complicated malaria. TH cells did not appear to be the source of increased IFN-γ expression associated with malaria severity.

Negative regulation of natural killer cell in tumor tissue and peripheral blood of oral squamous cell carcinoma.

Natural killer (NK) cells are the key lymphocytes in solid tumors. Its activity is regulated by both germline encoded receptors and cytokine microenvironment. We conducted a case-control study to investigate the activation status of NK cell in oral squamous cell carcinoma (OSCC). NK cell activation was assessed in context of NK cell cytotoxicity and transcript expression of NK cell receptors (NKp46 and KIRs) and NK cell associated cytokines (IL-1β, IL-2, IL-10, IL-12β, IL-15, IL-18, IL-21, IFN-γ, TNF-α and TGF-β). The results revealed possible mechanisms involved in reduced NK cell activation in peripheral circulation: quantitative deficiency of NK cell number and lowered cytotoxicity together with qualitative NK impairments caused by--(1) decreased expression of NK activating receptor NKp46, (2) increased expression of NK suppressive cytokines--IL-10 and TGF-β and (3) induction of FOXP3(+)CTLA4(+) suppressor cells. On the other hand, in the tumor tissue, escape of NK immune surveillance appeared to be modulated by upregulation of TGF-β and IL-10 together with downregulation of NK cell activating cytokines (IL-2, IL-12β, IL-15, IL-18, IL-21 and IFN-γ) and NK receptors (NKp46 and KIRs). In addition, our study supported the earlier contention that TNF-α and IL-1β expression levels may be used as markers of malignant transformation in oral leukoplakia. In conclusion, the study provided an insight into the negative regulation of NK cell in tumor tissue and peripheral blood of OSCC patients, which can be exploited to boost the current NK cell and cytokine based immunotherapy for the treatment of oral cancer.

Differential association of KIR gene loci to risk of malaria in ethnic groups of Assam, Northeast India.

Receptors encoded within the Natural Killer Cell (NKC) complex and Killer Immunoglobulin like (KIRs) genomic regions have been suggested to influence malaria pathogenesis and infection susceptibility. We have examined KIR locus in relation to risk of infection and disease in Tea tribes (TT) of Austro Asiatic affinity and Tibeto-Burman (TB) populations from malaria endemic regions of Assam. Consistent with differences in their genetic background, KIR gene loci frequencies differed in studied groups. Surprisingly, KIR3DS1 frequency in TT was low (17%) and comparable to that reported from African populations. KIR3DL1 frequency was positively associated with malaria severity (Pearson phi, R(2) = 0.297 p = 0.006) and logistic regression modelling predicted KIR3DL1 as a risk factor in complicated malaria [Odds Ratio (95% C.I)] = [6.39 (1.34-30.60)]. An interaction between ethnicity and KIR3DL1 was also seen where higher proportion of KIR3DL1 positive and complicated malaria patients belonged to Tea tribes (p = 0.009). Notably, four activating genes protected from frequent malaria (p = 0.02) while six activating genes enhanced the risk of complicated malaria (p = 0.05). Combination of KIR2DS4, KIR2DS4del, KIR2DS5 negatively influenced disease outcome in Tea tribes (p = 0.048) but not in Tibeto-Burman. In conclusion our data indicates KIR gene loci differentially influenced malaria outcome in Tea tribes and Tibeto-Burman and that four activating genes appeared to provide optimal activation that protected from frequent episodes of malaria. Our data also indicated KIR3DS1 to be an ancestral genotype, maintained at low frequency possibly by malaria in the Austro Asiatic tribes.

The association of IL-8-251T/A polymorphism with complicated malaria in Karbi Anglong district of Assam

Amongst host genetic factors, cytokine gene polymorphism can be anticipated to be an important factor as qualitative, quantitative and time of secretion play an important role in disease outcome. We have investigated association of cytokine promoter SNPs with risk of Plasmodium falciparum malaria and disease severity in a case control study in malaria endemic Karbi Anglong district of Assam, India. Frequency of IL-8-251T/A (p=0.03 and p=0.01) and TGF-β1-509C/T (p=0.02 and p=0.03) was higher in malaria in comparison to control participants and non-malarial fever controls. Interestingly, a higher frequency of mutant allele of IL-10-819T/C was observed in non-malarial fever controls compared to malaria thus suggesting its role as a distinguishing marker of the two disease groups. Higher IL-8 expression and increased frequency of IL-8-251T/A in complicated malaria (p=0.002) was reported indicating its role in susceptibility to complicated malaria. In conclusion, our study suggests the role of mutant genotype of IL-8-251T/A as a marker of complicated malaria in our population. Surprisingly, decreased expression of TGF-β1 in uncomplicated malaria even in presence of high expressing mutant genotype was observed and needs to be investigated in context of the pool of activated cells producing the cytokine.

Antibody response to allelic variants of 19kDa fragment of MSP-1: recognition of a variant and protection associated with ethnicity in Assam, India.

Evidence suggests association of anti MSP-1(19) antibodies with protection from clinical malaria. However, the target epitope was reported to vary with respect to response to conserved or variant epitopes in different studies. We have investigated here humoral response of naturally exposed individuals of Tibeto-Burman and Austro-Asiatic ethnic groups to E-TSR and Q-KNG variants of MSP-1(19) in comparison to whole merozoite extract (WME) of local strain of Plasmodium falciparum in a longitudinal prospective cohort study. The association of antibodies in relation to risk of infection and disease severity was determined. A relatively lower seropositivity to MSP1(19) peptides derived from 3D7 and FVO strains in comparison to whole merozoite extract of local P. falciparum strain was observed. Recognition of Q-KNG variant was markedly lower in TB (p<0.0001) indicating a role of ethnicity. The Tea tribes of Austro-Asiatic affinity had higher antibody response (E-TSR; p=0.038 and Q-KNG; p=0.004) and equally recognized the two variants. A reduced risk of clinical infection in high transmission summer season was seen in presence of anti MSP-1(19) antibodies (p=0.013) and antibody level was predictive of risk of clinical malaria (ROC=0.729). Anti E-TSR antibodies were inversely associated to disease severity at KTE (λ(2)p=0.013; t-test p=0.032). The present study demonstrated antibody response to MSP-1(19) was associated with protection from frequent episodes of malaria and disease severity and that the host genetic background was important factor in response to MSP-1(19) allelic variant.

KIR diversity in three ethnic populations of Assam state, Northeast India

Killer immunoglobulin-like receptors (KIR) exhibit extensive diversity and it has been observed that populations with different ethno-history, linguistic, geographic and genetic backgrounds can differ in KIR profile. In this context, we have investigated the KIR complex in three ethnic populations-Kachari (n = 108), Ahom (n = 104) and Adivasi (n = 101) of Assam, Northeast India. The three populations had 145 distinct KIR genotypes in 313 individuals typed. The two Mongoloid populations--Kachari and Ahom had close genetic affinities with their parental East Asian groups where the Kachari clustered with Chinese populations and the Ahom in another clade clustered with Thailand Bangkok and Polynesian populations. The Adivasi differed markedly from these Mongoloid populations in having higher KIR 2DL2, 2DS2, 2DS3 and 2DS5, but lower 2DL3 (P value <0.0001). Like the other native Indian populations, the Adivasi had higher share of Bx-haplogroup and C4Tx genotype (37/101). However, unlike other Indian populations, KIR 3DS1 gene frequency was lower in Adivasi (21%) and was comparable to the African populations. The neighbor-joining dendogram generated on the basis of KIR gene frequencies of our study populations with 43 world populations also placed the Adivasi with African populations. Interestingly, the three populations in the dendogram are consistent with their migration histories. In summary, our data suggest that KIR profile of the three ethnic populations displayed ethnic diversity and was consistent with their migration history thereby supporting the concept that KIR diversity may be used to understand genetic affinity and migration history of populations.

Integrative Approaches to Understand the Mastery in Manipulation of Host Cytokine Networks by Protozoan Parasites with Emphasis on Plasmodium and Leishmania Species

Diseases by protozoan pathogens pose a significant public health concern, particularly in tropical and subtropical countries, where these are responsible for significant morbidity and mortality. Protozoan pathogens tend to establish chronic infections underscoring their competence at subversion of host immune processes, an important component of disease pathogenesis and of their virulence. Modulation of cytokine and chemokine levels, their crosstalks and downstream signaling pathways, and thereby influencing recruitment and activation of immune cells is crucial to immune evasion and subversion. Many protozoans are now known to secrete effector molecules that actively modulate host immune transcriptome and bring about alterations in host epigenome to alter cytokine levels and signaling. The complexity of multi-dimensional events during interaction of hosts and protozoan parasites ranges from microscopic molecular levels to macroscopic ecological and epidemiological levels that includes disrupting metabolic pathways, cell cycle (Toxoplasma and Theileria sp.), respiratory burst, and antigen presentation (Leishmania spp.) to manipulation of signaling hubs. This requires an integrative systems biology approach to combine the knowledge from all these levels to identify the complex mechanisms of protozoan evolution via immune escape during host–parasite coevolution. Considering the diversity of protozoan parasites, in this review, we have focused on Leishmania and Plasmodium infections. Along with the biological understanding, we further elucidate the current efforts in generating, integrating, and modeling of multi-dimensional data to explain the modulation of cytokine networks by these two protozoan parasites to achieve their persistence in host via immune escape during host–parasite coevolution.