1,25-dihydroxyvitamin D3 augments low-dose PMA-based monocyte-to-macrophage differentiation in THP-1 cells.

The human monocytic THP-1 cell line is the most routinely employed in vitro model for studying monocyte-to-macrophage differentiation. Despite the wide use of this model, differentiation protocols using phorbol 12-myristate-13-acetate (PMA) or 1,25-dihydroxyvitamin D3 (1,25D3) vary drastically between studies. Given that differences in differentiation protocols have the potential to impact the characteristics of the macrophages produced, we aimed to assess the efficacy of three different THP-1 differentiation protocols by assessing changes in morphology and gene- and cell surface macrophage marker expression. THP-1 cells were differentiated with either 5 nM PMA, 10 nM 1,25D3, or a combination thereof, followed by a rest period. The results indicated that all three protocols significantly increased the expression of the macrophage markers, CD11b (p < 0.001) and CD14 (p < 0.010). Despite this, THP-1 cells exposed to 1,25D3 alone did not adopt the morphological and expression characteristics associated with macrophages. PMA was required to produce these characteristics, which were found to be more pronounced in the presence of 1,25D3. Both PMA- and PMA with 1,25D3-differentiated THP-1 cells were capable of M1 and M2 macrophage polarization, though the gene expression of polarization-associated markers was most pronounced in PMA with 1,25D3-differentiated THP-1 cells. Moreover, the combination of PMA with 1,25D3 appeared to support the process of commitment to a particular polarization state.

Lack of association of KIR2DL1-R245 and KIR2DL1-C245 with HIV-1 control in black South Africans with HLA-C2.

Activating/inhibitory Killer-cell Immunoglobulin-like Receptors (KIRs) partly regulate Natural Killer (NK) cells. KIR2DL1 allotypes with cysteine at position-245 (KIR2DL1-C245) express at lower levels and demonstrate weaker inhibitory signaling compared to allotypes with arginine at position-245 (KIR2DL1-R245). The functional consequence of either allotype in infectious diseases is unknown. Since NK cells mediate antiviral immunity, we investigated KIR2DL1-R245 and KIR2DL1-C245 in association with HIV-1 virological control in untreated immunocompetent black South Africans. Allotype carriage, determined by KIR2DL1 sequencing, was similar between uninfected South Africans (n = 104) and other black African populations, but differed significantly from Europeans, while no significant differences were noted between uninfected and HIV-1-infected individuals (n = 52). KIR2DL1 expression, measured by flow cytometry, in uninfected individuals showed higher KIR2DL1-R245 expression compared to KIR2DL1-C245 in white donors (n = 27), while black donors (n = 21) generally expressed lower levels of both allotypes. KIR2DL1 expression was reduced in HLA-C2 carriers, most evident in black HLA-C2/C2 donors. KIR2DL1-R245 and KIR2DL1-C245 did not associate with viral load when HLA-C2 ligands were present, however in HLA-C1 homozygotes, individuals with only KIR2DL1-R245, showed lower viral loads compared to carriers of both allotypes. The lack of association of KIR2DL1-R245 or KIR2DL1-C245 with HIV-1 control in HLA-C2 carriers may relate to lower KIR2DL1 expression levels in a population with high HLA-C2 prevalence.

Human leukocyte antigen class I (A, B, C) and class II (DPB1, DQB1, DRB1) allele and haplotype variation in Black South African individuals.

South Africa has a population of 58.78 million, of which 80.7% are Black African individuals, representing 9 predominant ethnic/linguistic groups (Zulu, Xhosa, Pedi, Tswana, South Sotho, Tsonga, Swati, Venda and Ndebele). HIV-1 and Mycobacterium tuberculosis infection are the leading causes of death (7.8% and 5.9%, respectively) in this population group. To provide reference HLA allele and haplotype data for studies of gene-associations with infectious/non-infectious diseases or vaccine development, we have updated previously published HLA class I (A, B, C) and class II DRB1 genotypes and determined high-resolution class II (DPB1, DQB1) genotypes for n = 142 healthy, unrelated Black South African individuals.

Killer-cell immunoglobulin-like receptor (KIR) and human leukocyte antigen (HLA) class I genetic diversity in four South African populations.

Killer-cell Immunoglobulin-like Receptor (KIR) and Human Leukocyte Antigen (HLA) genotypes vary considerably between individuals and populations due to KIR/HLA allelic variation and variable haplotype configurations of KIR. HLA mediate natural killer cell activity by serving as KIR ligands. KIR/HLA polymorphisms associate with both disease susceptibility and severity. We determined the frequencies of KIR, KIR genotypes and KIR-HLA combinations in 364 healthy individuals from four South African populations. Study participants included black African (n=167), Caucasian (n=97), Mixed ancestry (n=50) and Indian (n=50) individuals. We identified 48 KIR genotypes that included two genotypes not previously reported. Based on KIR gene content, Indian individuals represented the most distinct group, showing the highest frequencies of KIR2DL2, KIR2DL5, KIR2DS1, KIR2DS2, KIR2DS3 and KIR3DS1, the lowest frequencies of KIR2DL3, KIR2DS4 and KIR3DL1; and a KIR2DL4-negative individual. KIR2DS1 and KIR3DS1 were infrequent in black African populations. HLA-C2 was more common in black African individuals, while HLA-C1 predominated in the other populations. Indian individuals were more likely to possess KIR2DL2 paired with HLA-C1, while Caucasian individuals exhibited the highest frequencies of KIR2DL3 paired with HLA-C1. This report provides comprehensive reference data for further study of the roles of KIR/HLA in non-communicable and infectious diseases in South African populations.

Genetic variability in markers of HLA-C expression in two diverse South African populations.

An insertion-deletion (indel) polymorphism within the 3' untranslated region (UTR) of HLA-C has been shown to be involved in the regulation of HLA-C expression. Individuals who carry a deletion at this position exhibit increased HLA-C expression, which associates with lower viral set point in HIV-1 infected individuals. This 263 indel (rs67384697) is reported to be in strong linkage disequilibrium (LD) with a single nucleotide polymorphism (SNP) 35 kilobases upstream of HLA-C (-35T/C; rs9264942) in Caucasian individuals, making this SNP a potential marker for both HLA-C expression and HIV-1 disease progression. We therefore examined genetic variation within the HLA-C 3' UTR of 265 Black and Caucasian South Africans by direct sequencing and identified haplotypes encompassing the 263 indel and another indel at position 230 in both populations. Concomitant evaluation of variability at the -35 SNP revealed this polymorphism to be an inappropriate marker for the 263 indel in these populations. These findings provide important insights into genetic variability within the regulatory regions of HLA-C that have potential implications for our understanding of the regulation of HLA-C expression and its impact on HIV-1 disease progression.

Human leukocyte antigen class I (A, B, C) and II (DRB1) diversity in the black and Caucasian South African population.

A cross-section of black and Caucasian South Africans (N = 302) were genotyped at high resolution (class I HLA-A, -B, -C and class II HLA-DRB1). Five new class I alleles (A*30:01:02, A*30:02:02, A*68:27, B*42:06, and B*45:07) and one new confirmatory allele (A*29:11) were identified in the black population. Alleles and haplotypes showed expected differences between the black and Caucasian populations, with the black population, on average, showing a broader spectrum of allele representation (less single allele dominance). The most prevalent alleles at the four loci in the black population were A*30:01, B*58:02, C*06:02, and DRB1*13:01 and in the Caucasian population were A*02:01:01, B*07:02:01, C*07:01, and DRB1*03:01. HLA-B, and HLA-C loci showed the strongest overall linkage disequilibrium (LD) and HLA-B/HLA-C two locus haplotypes also showed the strongest LD (D'(ij)) in both population groups. Bw allotype representation was similar between the two populations; however C allotypes differed significantly (C1 higher representation in Caucasians; C2 higher representation in blacks). HLA-A Supertype family phenotypic frequencies did not differ between the two populations, but four (B08, B27, B58, and B62) HLA-B Supertype families differed significantly. However, vaccine coverage estimation came close to 100% in both population groups, with inclusion of only four Supertype families (A1, A2, B7, B58).