Browsing by Author "Chigorimbo-Tsikiwa, Nyaradzo"
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- ItemOpen AccessCharacterization of human foreskin Langerhans cells(2021) Qumbelo, Yamkela; Chigorimbo-Tsikiwa, Nyaradzo; Gray, CliveBackground: It is known that medical male circumcision (MMC) decreases HIV acquisition by up to 60%. One hypothesis is that MMC removes a foreskin (FS) that harbors different immune cells that are HIV target cells such as CD4+ macrophages, T, Langerhans (LCs), and dendritic cells (DCs). However, there have been different reports on whether the inner FS or outer FS has more HIV target cells. While LCs have been implicated in HIV transmission, their role remains controversial. Studies have shown that LCs can transmit the virus to T cells, which increases infection. On the contrary, others have reported that LCs prevent infection by degrading the virus through a langerin-dependent pathway. One of the factors that plays a major role in HIV transmission is their state of maturity and activation, which can be influenced by co-infection and other immunological processes. The aim of this study was to isolate, quantify and characterize Langerhans cells in the inner FS and outer FS from men undergoing MMC and to evaluate the phenotype of matured and activated LCS. Differences in the proteome of the inner FS and outer FS tissues were further investigated. Methodology: FS were obtained from men undergoing voluntary MMC from clinics and hospitals in the Western Cape (Age 18 years or older). Epidermal FS cells were extracted using crawl (migratory) assay and liberase enzyme digestion. Langerhans cells were isolated by density gradient centrifugation, sorted, quantified and immune-profiled by flow cytometry. CD1a and CD207 were used to identify Langerhans cells while HLA-DR, CD80, CD86 and CD40 were used as markers of maturity and activation. The gene expression profile of sorted LCs was also examined by single-cell sequencing with seq-well. Lastly, the differences in the proteome of the inner FS and the outer FS migrated epidermal cells were assessed using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Results: Langerhans cells were an average of 85% pure post-sorting. The numbers of Langerhans cells between the inner FS vs. outer FS were not statistically different (mean: 0.56% vs. 0.68% (SD=0.37) from migratory cells and 0.28% vs. 0.45% (SD=0.18) from enzyme digest, p-value >0.05, n=9). Sequencing showed that the sorted cells pooled from 5 participants (inner and outer FS) had different gene expression profiles. Furthermore, two groups of cells were identified from the sorted LCs based on their gene expression profile. The identified cells were monocyte-like and melanocyte-like cells. The monocyte-like cells were identified as LCs based on their gene expression profile while the melanocyte-like cells were identified as the contaminating cells as the cell purity was not 100%. Upon activation with tumor necrosis factor alpha (TNF-α), activated LCs isolated by the migration assay had similar proportions of cells expressing surface maturity and activation markers (HLA-DR, CD40 and CD80/86) when compared to the unstimulated controls (inactivated) (mean: 73.53% vs. 75.66%, n=9, p-value >0.05, SD=4.4) However LCs that were isolated by the migration assay expressed markers of activation at a higher level compared to LCs isolated by Liberase enzyme digestion (mean: 79.4% vs. 40%, p-value < 0.05 n=9, SD=23). Proteomics showed that the inner FS had an over-abundance of proteins involved in the interleukin 7 response and mRNA catabolic processes, while the outer FS had more spindle zones and cornified envelope proteins that were over-abundant when comparing inner and outer FS from 5 participants. Discussion and Conclusion: The study successfully extracted, sorted and immunoprofiled Langerhans cells using different methods and from different FS compartments (inner FS versus outer FS). When LCs were spontaneously migrated and isolated using the “crawl method”, they showed a more mature and activated phenotype compared to non-migrating “skin resident” immune cells. No differences were found between cells that were stimulated with inflammatory cytokines relative to unstimulated migratory cells in proportion of cells expressing activation markers. However, it was observed that cells isolated by liberase enzyme digestion showed significantly lower proportions of activation markers relative to migratory cells. Using LC-MS/MS-based proteomics; the inner FS exhibited high expression of proteins involved in the interleukin 7 response while the outer FS exhibited high expression of structural proteins, which suggests that the inner FS might be more involved in immunity as interleukins can stimulate immune response while the outer FS has a more structural role than the inner FS.
- ItemOpen AccessInvestigating the susceptibility of foreskin myeloid cells to ex vivo HIV infection(2023) Nleya, Bokani; Chigorimbo-Tsikiwa, Nyaradzo; Dzanibe SonwabileBackground: HIV/AIDS remains a global concern that, although manageable using anti-retroviral therapy (ART), is still eluded by a cure with paucity of knowledge regarding its acquisition and spread especially through the male genital tract (MGT)1–4. Several authors have shown the human foreskin to be an effective mucosal effector site with heterogenous populations of innate and adaptive immune cells, that are permissive to HIV infection5–8. In support of this, medical male circumcision (MMC), has been reported to confer up to 60 % risk reduction in HIV acquisition9–17. Most studies have focused on investigating blood lymphoid immune cells and their interaction with HIV-1, this study sought to elucidate the myeloid cell composition of the inner and outer foreskin, and to investigate the susceptibility of these cells to ex vivo HIV infection by (i) Isolating migratory and non-migratory Langerhans cells (LCs) and “macrophage-like” cells from the foreskin epidermis (ii) Immunophenotyping and characterising foreskin LCs and “macrophage-like” cells using CD4+CCR5+ as proxy for HIV susceptibility, HLA-DR+CD80/86+ for maturation, and the mannose receptor, DC-SIGN and Siglec-1 as HIV attachment factors and (iii) Investigating the HIV susceptibility of foreskin epidermal cells using an optimised ex vivo pluricellular foreskin infection model of suspension cells. Methodology: Foreskin specimen were obtained from 60 seronegative adult South African men (aged 18-35 years) undergoing voluntary medical male circumcision (vMMC). Migratory and non-migratory foreskin cells were isolated from the inner and outer foreskin using spontaneous migration and enzymatic digestion of remnant epidermal tissue respectively, and subsequently immunophenotyped using multiparameter flow cytometry (n=31). The optimal HIV infection model was determined through assessment of different infection models inclusive of i) epidermal sheets, ii) foreskin explants and iii) pluricellular suspension cells (n=5). Using the ex vivo pluricellular foreskin infection model of suspension cells (n=17), Subtype C transmitted founder (T/F) and chronic infection derived (CC) infectious molecular clones (IMCs) were used alongside Subtype B NL4-3 IMCs with CCR5, CXCR4 and BaL envelopes. The extent of HIV infection was quantified by measurement of p24 in different immune cell subsets over a time-course. The different HIV infected cell subsets were characterized using CD45, CD207, CD1a, CD11c, CD14, CD3, HLA-DR, CD80/86, CD209, CD206, CD169, CD4 and CCR5. Results: Foreskin myeloid cells contained a rare population of LCs (1.11 % ± 1.02 %;) that was predominantly migratory (p = 0.0084) and “macrophage-like” cells (9.87 % ± 9.64 %) that, in addition to being 8-fold more abundant (p
- ItemOpen AccessUsing immunofluorescence techniques to Identify T cells in the foreskin tissue after medical male circumcision(2022) Sebaa, Shorok; Gray, Clive; Chigorimbo-Tsikiwa, NyaradzoBackground: Medical Male Circumcision (MMC) plays an important role in reducing the risk of acquiring sexually transmitted infections (STIs) such as Human papilloma virus (HPV), Herpes simplex type 2 (HSV-2) and HIV-1. The foreskin tissue (FS) is a site abundant in Langerhans cells (LCs), macrophages and T helper cells that express CD4 and CCR5 that are target markers for HIV1 binding and viral infection. The foreskin tissue may also contribute chemokines and cytokines including those that promote inflammation such as IL-17, IL-1β, IL-8, MCP-1 and MIG. The inner foreskin has been shown to contain higher levels of CD4+CCR5+ cells and thus more susceptible to HIV infection compared to the outer foreskin. It was demonstrated that the majority of chemokines measured were highly expressed in the inner foreskin compared to the outer foreskin including CCL27 which was approximately 7-fold higher in the inner foreskin compared to the outer foreskin, in congruent with the higher density of CD4+CCR5+ observed in the epithelium of the inner foreskin. In this study, we hypothesized that CCL27 upregulation in the inner foreskin triggers the recruitment of CD4+ T cells to the epithelium of the foreskin tissue. This could subsequently lead to increased susceptibility to infections in the inner foreskin tissue. The aims of this dissertation were: 1) to measure the impact of CCL27 on the recruitment of CD4+ T cells to the epithelium of the foreskin tissue using immunofluorescence imaging. 2) to compare manual counting and semi-automated method for counting dually positive cells. 3) to use multiparameter flow cytometry to characterize the cells recruited under the influence of CCL27. Methodology: Inner foreskin tissue (n=11) and outer foreskin tissue (n=4) explants were treated with either TNFα or CCL27 and evaluated using immunofluorescence imaging to quantify the levels of CD3 and CD4 expressing cells. Dually positive CD3+CD4+ cells were counted manually using softworx software on the Deltavision microscope and with semi-automated counting using PIPSQUEAK on ImageJ. TNFα and CCL27 treated inner and outer FS cells were immunophenotyped using polychromatic flow cytometry to measure and compare the densities of Th17 and Th22 cells under the influence of the chemokines. Results: Exogenous exposure of inner foreskin tissue explants to TNFα showed a significant increase in the median density of CD3+CD4+ T cells in the epithelium of the inner foreskin (p=0.035) from 78.90 cells/mm2 (IQR: 33.02-127.50) in the unstimulated inner foreskin explants to 134.80 cells/mm2 (IQR: 109.30-206.60). Similarly, the addition of exogenous CCL27 resulted in the median density of CD3+CD4+ T cells in the epithelium of the inner foreskin to increase from the unstimulated inner foreskin (value above) to 164.80 cells/mm2 (IQR: 140.30-184.90, p=0.008). No significant difference was observed in the median density of CD3+CD4+ T cells in the outer foreskin tissue explants after exposure to TNFα and CCL27 (36.50 cells/mm2 , IQR: 18.29-96.65 in the unstimulated tissues compared to 65.12 cells/mm2 , IQR: 7.30-202.80 in the TNFα stimulated tissues; p>0.999 and 24 cells/mm2 , IQR: 11.35-149.40 for the CCL27 stimulated tissues; p=0.686). The median density of CD3+CD4+ T cells in the epithelium of unstimulated inner foreskin tissue showed a trend of an increase from the unstimulated outer foreskin tissue but was not statistically different (127.50 cells/mm2 , IQR: 89.22-219.50 in the inner foreskin compared to 36.52 cells/mm2 , IQR:18.29-96.65 in the outer foreskin explants; p=0.057). When comparing the cell counting methods: manual counting vs semi-automated counting, we observed that the manual counting method estimated higher numbers of dually positive cells compared to the semiautomated method in samples measuring 200 cells/mm2 . Despite these differences, there was strong correlation (R=0.782, p0.999) in CCL27 treated explants. The median frequency of Th22 cells in the inner foreskin in the unstimulated tissue explants was 8.80% (IQR: 1.68-12.60%) vs 5.30% (IQR: 0.96-7.67%, p=0.250) in TNFα treated explants and 4.90% (IQR:0.75-7.39%, P=0.125) in CCL27 treated explants. Meanwhile, the median frequency of Th17 cells in the outer foreskin in the unstimulated tissue explants was 21.60% (IQR: 15.40-37.33%) vs 28.20% (IQR: 14.60-39.40%, P=0.750) in TNFα treated explants and 22.90% (IQR:22.90-29.50%, p>0.999) in CCL27 treated explants. The median frequency of Th22 cells in the outer foreskin in the unstimulated tissues was 4.67% (IQR: 2.30-12.90%) vs 5.37% (IQR: 5.34- 7.58%, P=0.750) in TNFα treated tissues and 4.45% (IQR:3.64-5.98%, p>0.999) in CCL27 treated tissues. Furthermore, FS cells isolated using Dispase had significantly lower median frequencies of cells expressing CCR6 (18.35%, IQR:1.33-28.30%) compared to whole tissue controls (41.90%, IQR: 22.46-67%, p=0.031). This impacted the characterization of CD4+ T cell subsets in FS cells and limited our ability to adequately phenotype and measure the impact of TNFα and CCL27 on FS-derived cells using flow cytometry. Conclusion: This study demonstrates that exogenous exposure of FS to TNFα and CCL27 increased the density of CD3+CD4+ T cells in the epithelium of the inner but not the outer foreskin tissue. It was noteworthy that the density of CD3+CD4+ in the epithelium of the inner foreskin was higher than the outer in the unstimulated tissues, suggesting that the proinflammatory environment in the inner FS potentially leads to higher density of T cells in the inner FS even without exogenous stimulation. These results suggest a possible mechanism for recruiting HIV target cells in the inner foreskin tissue associated with higher levels of CCL27 that recruits HIV-1 target T cells during inflammatory responses. A limitation to this conclusion is the small sample size in the outer foreskin. The study also shows potential bias depending on the method used to quantify dually positive cells, whereby semi-automated counting underestimated the densities of CD3+CD4+ T cells compared to manual counting and therefore careful consideration is required when selecting the quantification method. Furthermore, there were no significant difference in the frequencies of Th17 and Th22 cells after exposure to TNFα and CCL27 using flow cytometry. The effects of Dispase on cell surface marker expression and the low cell yield across the experiments impacted the characterization of Th17 and Th22 using flow cytometry and thus limiting capacity to determine how CCL27 influences these T cell subsets.