Natural killer (NK) cells are important in the early control of viral infections and genetic evidence and in-vitro studies suggest a potential role for NK cells in controlling HIV-1 infection [1–3]. NK cells are not normally able to prevent infection by HIV-1, however, and once established, HIV-1 drives NK cell depletion in the peripheral blood and a shift in blood NK cell populations towards a functionally compromised CD56 negative NK cell population and an NKG2C+ subset with altered NK receptor expression [4–7]. Control of plasma viraemia, using highly active antiretroviral therapy (HAART), leads to a reversal of some effects of chronic HIV-1 infection on peripheral NK cells, including repopulation and a reduction in the proportion of CD56− NK cells in the blood .
During acute HIV-1 and simian immunodeficiency virus (SIV) infection there is a rapid decline in the CD4+ T-cell population in the gut with parallel effects on the architecture of lymph nodes in the gastrointestinal tract [8–12]. These changes persist during chronic infection and there is only limited recovery of CD4+ T cells in the gastrointestinal tract after antiretroviral therapy [13–15]. We therefore investigated the impact of HIV-1 infection on NK cells in colonic lamina propria (LPNK cells) compared to the blood (PBNK cells) of HIV-1-infected individuals with detectable plasma virus or after viral suppression with antiretroviral therapy.
Patients and sample acquisition
All patients were recruited from the endoscopy department of the Chelsea and Westminster Hospital. Endoscopic examination and biopsy of the large intestine was carried out according to the clinical need of the patients. Informed consent for all study procedures was obtained in all patients. Twenty-seven HIV-1-positive individuals with plasma viral load below the limit of detection (50 copies/ml), 15 HIV-1-positive individuals with detectable plasma viraemia and 26 HIV-1-negative control individuals were recruited The median age for HIV-1-negative control patients was 47 years (range, 28–71 years). Patient characteristics are shown in Table 1.
Biopsies were taken distant from any discreet pathology such as ulcers or polyps. Both HIV-1-positive and HIV-1-negative control patients were selected from patients attending for endoscopic examination because of: personal history of colonic polyps, familial history of polyposis or for investigation of rectal bleeding. All HIV-1-negative controls had no overt pathology on endoscopic examination.
Biopsies for evaluation of LPNK populations were taken from the sigmoid colon (×6) circumferentially at 30 cm from the anus. The biopsy forceps used were Olympus FB 242 radial jaw with external diameter of 3.7 mm (Keymed, Southend-on-Sea, UK). Parallel blood samples were taken into lithium heparin vacutainers (Becton Dickinson, Oxford, UK) and peripheral blood mononuclear cells were separated by density gradient centrifugation on Histopaque-1077 (Sigma, Poole, UK.)
Processing of biopsies
Biopsies obtained during colonoscopy were collected in ice-cold Dutch modification of RPMI 1640 supplemented with antibiotics. Epithelium and associated lymphocytes were removed by repeated treatment with ethylenediamine tetra-acetic acid and washing and the lamina propria lymphocytes (LPL) were prepared as described previously . Briefly, for phenotypic analysis, epithelium and intra epithelial lymphocyte (IEL)-free tissue was digested with 1 mg/ml collagenase D (Roche Molecular Products, Basel, Switzerland) in a Dutch modification of RPMI 1640 containing 20 mg/ml DNase I (Roche Molecular Products) and 2% fetal calf serum to release lamina propria lymphocytes.
Flow cytometric analysis
Flow cytometry was used to reliably estimate the proportion of NK cells within leukocyte populations extracted from peripheral blood and from colonic biopsies and discriminating between NK cells and CD3+ T-cell populations bearing NK cell markers (see Fig. 1a). Leukocytes from tissue and peripheral blood were identified by staining with fluorescein conjugated anti-CD45 (Sigma Aldrich, Poole, UK) and peridinin-chlorophyll protein (PercP) conjugated anti-CD3 (BD Biosciences, Oxford, UK) either in combination with Allophycocyanin (APC)-conjugated CD56 and phycoerythrin (PE)-conjugated CD16 (Both from Beckman Coulter, Abingdon, UK) to identify NK cell populations, or in combination with anti-CD8 APC (Beckman Coulter) and anti-CD4 PE (BD Biosciences) to identify CD3+CD8+ and CD3+CD4+ T-cell populations, respectively. Flow cytometry was performed using FacsCalibur flow cytometer (Becton Dickinson) and a minimum of 20 000 events were acquired after gating on small lymphocytes according to CD45 and side scatter (Fig. 1a).
Comparisons of cell populations between patient groups were made using the Mann–Whitney U test. StatView 5.01 software was used (Abacus, Berkley, California, USA) and P values < 0.05 were considered as significant.
Lower proportion of natural killer cells in viraemic HIV-1-positive individuals but not in aviraemic individuals receiving HAART
The proportions of CD3+CD56+ NK cells were analysed in gated CD45+ lymphocyte fractions from colonic biopsies and peripheral blood of HIV-1-positive individuals with detectable plasma viraemia (viraemic), HIV-1-positive individuals without detectable plasma viraemia (aviraemic) and HIV-1-negative control individuals (Fig. 1a). No difference was observed in the proportions of total CD45+ lymphocytes in cells extracted from colonic biopsies between treatment HIV-1-positive aviraemic (mean = 74+/−23%), viraemic individuals (mean = 74+/−20%) and HIV-1 negative control individuals (mean = 64.4+/−31.1%) (data not shown).
The median and range of absolute blood CD4+ T-cell counts in aviraemic individuals was similar to that observed in viraemic individuals (Table 1). We made use of this similarity to study the impact of plasma viraemia on the proportion of NK cells and T-cells in the blood and the gut of infected individuals.
Analysis of NK cells from both peripheral blood and lamina propria focused on CD56+CD16+ and CD56+CD16− populations as CD56negativeCD16+ NK cells were entirely absent from lamina propria lymphocytes (data not shown). A decrease in the proportion of CD3−CD56+ LPNK cells was detected within gated CD45+ lymphocytes from colonic lamina propria of viraemic HIV-1-infected individuals (median = 3.0, range = 0.6–9.0) in comparison with HIV-1-negative individuals (median = 4.8%, range = 1.3–17.4%, P = 0.035)(Fig. 1b). The median proportion of LPNK cells in aviraemic individuals (median = 5.3%, range = 1.4–17.6%) was similar to that observed for HIV-1-negative control individuals and was higher than in viraemic HIV-1-positive individuals, although not reaching significance (P = 0.125). NK cells also decreased in proportion in the blood of viraemic HIV-1-infected individuals (median = 3.6, range = 1.1–12.2%) in comparison with both aviraemic individuals (median = 8.4%, range = 1.8–20%, P = 0.018) and HIV-1-negative control individuals (median = 8.8%, range = 2.2–18.2%, P = 0.03), consistent with changes in the absolute numbers of CD16+CD56+ NK cells (Table 1) and with previous reports and indicating a recovery in the proportion of PBNK cells with viral suppression (Fig. 1b).
No difference was observed in the proportion of total CD3+ T cells within CD45+ leukocytes extracted from colonic lamina propria of HIV-1-positive viraemic individuals compared to aviraemic individuals and HIV-1-negative control individuals (Fig. 1d). A lower proportion of lamina propria CD3+CD4+ T cells was observed in viraemic HIV-1-positive individuals (median = 19.5%, range = 9.0–46.3%) compared to both aviraemic individuals (median = 44.8%, range 21.4–60.0%, P = 0.0022) and HIV-1-negative control individuals(median = 68.2%, range = 49.8–79.2%, P = 0.0008) (Fig. 1e). Conversely, viraemic individuals had a higher proportion of lamina propria CD3+CD8+ T cells (median = 71.4%, range = 16.7–87.1%,) in comparison with HIV-1-negative control individuals (median = 17.2%, range = 12.8–26.2%, P = 0.0032) and a higher median proportion in comparison with aviraemic individuals (median = 42.3%, range = 25.1–74%, P = 0.058) (Fig. 1f). The CD4/CD8 T-cell ratio in the colonic lamina propria was higher in aviraemic HIV-1-infected individuals (median = 1.07, range = 0.29–2.39) in comparison with viraemic individuals (median = 0.67, range = 0.11–1.21, P = 0.03) but did not approach that observed for HIV-1-negative controls (median = 4.02, range = 1.9–5.3), indicating that only a partial recovery of CD4+ T cells occurs on successful antiretroviral therapy (data not shown).
A lower proportion of peripheral blood CD4+ T cells was observed in both viraemic and aviraemic HIV-1-positive individuals in comparison with HIV-1-uninfected control individuals(median = 59.6%, range = 44.3–88.2%, P = 0.0003 and P = 0.0001, respectively) coincident with a reciprocal increase in the proportion of CD8+ T cells (median = 33.6%, range = 9.4–40.5%, P < 0.0002 and P < 0.0003, respectively) (Fig. 1e and f). Consistent with the absolute blood CD4+ T-cell counts, however, no significant difference was observed in the proportion of CD4 and CD8 T cells in the blood of viraemic (medians, ranges 29%, 12.7–49.5% and 59.3%, 40.6–78.5%, respectively) and aviraemic HIV-1-positive individuals (medians, ranges 34.6%, 9.7–60% and 56.7%, 6.6–85.8%, respectively)(Fig. 1e and f).
These results demonstrate a reduction in the proportion of NK cells and CD4+ T cells in colonic lamina propria of individuals with detectable plasma viraemia and support a role for viral suppression by antiretroviral therapy in the partial recovery of these subsets in chronically infected HIV-1-infected individuals.
Redistribution of natural killer subsets
The majority of PBNK cells are CD56loCD16+ in phenotype, express perforin and demonstrate cytolytic activity. CD56+CD16−PBNK cells are more effective producers of cytokines on a per cell basis. Differences in the distribution of these two subsets have been observed in HIV-1-infected individuals in comparison with HIV-1-seronegative individuals, CD16+ NK cells being lost in viraemic patients, likely due to the emergence of a CD16−NK cell population [17,18]. Colonic LPNK cells had a predominantly CD56+CD16− phenotype in both HIV-1-positive and HIV-1-negative individuals (Fig. 1c). An increased proportion of CD16+ LPNK cells was observed in both viraemic (median = 21.4%, range = 7.9–64.4%) and aviraemic (median = 21.1%, range = 2.9–60.3%) HIV-1-positive individuals, however, in comparison with HIV-1-negative control individuals (median = 6.3%, range = 0.1%–50.0%, P = 0.0046 and P = 0.0015, respectively) (Fig. 1c). These results demonstrate that NK cell subsets differ in the blood and lamina propria and that LPNK cell subset distribution is altered as a result of HIV-1 infection.
Discussion and conclusions
These studies demonstrate for the first time that a parallel depletion of NK cells and CD4+ T cells occurs in colonic lamina propria of HIV-1-infected individuals. Furthermore, recovery of NK and CD4+ T cells in colonic mucosal biopsy tissue is more effective in individuals with complete viral suppression in comparison with those with detectable plasma viral load. Lymphocyte reconstitution in the gastrointestinal tract is complex, with previous studies reporting delayed and only partial recovery of both colonic and jejunal CD4+ T cells in comparison with peripheral blood CD4+ T cells in individuals treated during chronic infection [13–15].
There is some evidence that HAART-induced CD4+ T-cell reconstitution is more effective in inductive lymphoid tissue than in lamina propria effector tissue from sigmoid colon, raising the possibility that effective HAART could lead to a more dramatic repopulation of NK cells at these sites . Plasma HIV-1 viral load correlates well with the detection of HIV-1 mRNA in mucosal mononuclear cells using quantitative reverse transcriptase polymerase chain reaction . The lack of colonic LPNK cell recovery in individuals with detectable plasma virus shown here may therefore reflect the persistence of virus production in these tissues.
Gross differences in the phenotype of LPNK and PBNK cells suggest specialized function of NK cells in mucosal tissue as has been demonstrated for other diverse tissues [20–24]. CD56+CD16− LPNK cells from HIV-1-negative and HIV-1-positive individuals produce both interferon-γ and tumor necrosis factor-α on in-vitro stimulation (data not shown), consistent with cytokine production in mucosal T cells from HIV-1-infected individuals . LPNK cells, however, contained few CD56−CD16+, NKG2C+ or perforin-expressing cells, which are all features of CD16+ peripheral blood NK cells from chronically infected HIV-1-positive individuals (data not shown). This is consistent with the detection of perforin-positive CD3+ and CD3− lymphocytes in mucosal biopsy tissue during acute but not during chronic HIV-1 or SIV infection [19,26]. An increased proportion of CD16+ LPNK cells in HIV-1-infected individuals may result from different pathogenic effects of chronic infection. Loss of tissue integrity in the gastrointestinal tract may cause an influx of blood CD16+ NK cells, or an increased burden of microbial products could lead to activation of CD56+CD16− LPNK cells and the acquisition of CD16 .
Uterine decidual NK (dNK) cells produce factors driving cellular growth and development in the uterus . Further studies will elucidate whether LPNK, which are phenotypically similar to dNK, have a similar role in growth and regeneration in the gastrointestinal tract. The extent of recovery of NK cell function will be important in evaluation of the benefits of antiretroviral therapy in the gastrointestinal tract of HIV-1-infected individuals.
This work was supported by grants from the St Stephens AIDS trust and Joint Research Committee of the Trustees of Chelsea and Westminster Hospital.
1. Bonaparte MI, Barker E. Killing of human immunodeficiency virus-infected primary T-cell blasts by autologous natural killer cells is dependent on the ability of the virus to alter the expression of major histocompatibility complex class I molecules. Blood 2004; 104:2087–2094.
2. Kottilil S, Chun TW, Moir S, Liu S, McLaughlin M, Hallahan CW, et al
. Innate immunity in human immunodeficiency virus infection: effect of viremia on natural killer cell function. J Infect Dis 2003; 187:1038–1045.
3. Martin MP, Gao X, Lee JH, Nelson GW, Detels R, Goedert JJ, et al
. Epistatic interaction between KIR3DS1 and HLA-B delays the progression to AIDS. Nat Genet 2002; 31:429–434.
4. Mavilio D, Lombardo G, Benjamin J, Kim D, Follman D, Marcenaro E, et al
. Characterization of CD56−/CD16+ natural killer (NK) cells: a highly dysfunctional NK subset expanded in HIV-infected viremic individuals. Proc Natl Acad Sci U S A 2005; 102:2886–2891.
5. Mela CM, Burton CT, Imami N, Nelson M, Steel A, Gazzard BG, et al
. Switch from inhibitory to activating NKG2 receptor expression in HIV-1 infection: lack of reversion with highly active antiretroviral therapy. AIDS 2005; 19:1761–1769.
6. Mavilio D, Lombardo G, Kinter A, Fogli M, La Sala A, Ortolano S, et al
. Characterization of the defective interaction between a subset of natural killer cells and dendritic cells in HIV-1 infection. J Exp Med 2006; 203:2339–2350.
7. Goodier MR, Mela CM, Steel A, Gazzard B, Bower M, Gotch F. NKG2C+ NK cells are enriched in AIDS patients with advanced Kaposi's sarcoma disease stage. J Virol 2007; 83:430–433.
8. Brenchley JM, Schacker TW, Ruff LE, Price DA, Taylor JH, Beilman GJ, et al
. CD4+ T cell depletion during all stages of HIV disease occurs predominantly in the gastrointestinal tract. J Exp Med 2004; 200:749–759.
9. Mehandru S, Poles MA, Tenner-Racz K, Horowitz A, Hurley A, Hogan C, et al
. Primary HIV-1 infection is associated with preferential depletion of CD4+ T lymphocytes from effector sites in the gastrointestinal tract. J Exp Med 2004; 200:761–770.
10. Li Q, Duan L, Estes JD, Ma ZM, Rourke T, Wang Y, et al
. Peak SIV replication in resting memory CD4+ T cells depletes gut lamina propria CD4+ T cells. Nature 2005; 434:1148–1152.
11. Mattapallil JJ, Douek DC, Hill B, Nishimura Y, Martin M, Roederer M. Massive infection and loss of memory CD4+ T cells in multiple tissues during acute SIV infection. Nature 2005; 434:1093–1097.
12. Centlivre M, Sala M, Wain-Hobson S, Berkhout B. In HIV-1 pathogenesis the die is cast during primary infection. AIDS 2007; 21:1–11.
13. Guadalupe M, Reay E, Sankaran S, Prindiville T, Flamm J, McNeil A, Dandekar S. Severe CD4+ T-cell depletion in gut lymphoid tissue during primary human immunodeficiency virus type 1 infection and substantial delay in restoration following highly active antiretroviral therapy. J Virol 2003; 77:11708–11717.
14. Guadalupe M, Sankaran S, George MD, Reay E, Verhoeven D, Shacklett BL, et al
. Viral suppression and immune restoration in the gastrointestinal mucosa of human immunodeficiency virus type 1-infected patients initiating therapy during primary or chronic infection. J Virol 2006; 80:8236–8247.
15. Mehandru S, Poles MA, Tenner-Racz K, Jean-Pierre P, Manuelli V, Lopez P, et al
. Lack of mucosal immune reconstitution during prolonged treatment of acute and early HIV-1 infection. PLoS Med 2006; 3:e484.
16. Hart AL, Al-Hassi HO, Rigby RJ, Bell SJ, Emmanuel AV, Knight SC, et al
. Characteristics of intestinal dendritic cells in inflammatory bowel diseases. Gastroenterology 2005; 129:50–65.
17. Tarazona R, Casado JG, Delarosa O, Torre-Cisneros J, Villanueva JL, Sanchez B, et al
. Selective depletion of CD56(dim) NK cell subsets and maintenance of CD56(bright) NK cells in treatment-naive HIV-1-seropositive individuals. J Clin Immunol 2002; 22:176–183.
18. Mavilio D, Benjamin J, Daucher M, Lombardo G, Kottilil S, Planta MA, et al
. Natural killer cells in HIV-1 infection: dichotomous effects of viremia on inhibitory and activating receptors and their functional correlates. Proc Natl Acad Sci U S A 2003; 100:15011–15016.
19. Mehandru S, Poles MA, Tenner-Racz K, Manuelli V, Jean-Pierre P, Lopez P, et al
. Mechanisms of gastrointestinal CD4+ T cell depletion during acute and early HIV-1 infection. J Virol 2007; 81:599–612.
20. Dalbeth N, Callan MF. A subset of natural killer cells is greatly expanded within inflamed joints. Arthritis Rheum 2002; 46:1763–1772.
21. Fehniger TA, Cooper MA, Nuovo GJ, Cella M, Facchetti F, Colonna M, Caligiuri MA. CD56bright natural killer cells are present in human lymph nodes and are activated by T cell-derived IL-2: a potential new link between adaptive and innate immunity. Blood 2003; 101:3052–3057.
22. Ferlazzo G, Thomas D, Lin SL, Goodman K, Morandi B, Muller WA, et al
. The abundant NK cells in human secondary lymphoid tissues require activation to express killer cell Ig-like receptors and become cytolytic. J Immunol 2004; 172:1455–1462.
23. Schierloh P, Yokobori N, Aleman M, Musella RM, Beigier-Bompadre M, Saab MA, et al
. Increased susceptibility to apoptosis of CD56dimCD16+ NK cells induces the enrichment of IFN-gamma-producing CD56bright cells in tuberculous pleurisy. J Immunol 2005; 175:6852–6860.
24. Hanna J, Goldman-Wohl D, Hamani Y, Avraham I, Greenfield C, Natanson-Yaron S, et al
. Decidual NK cells regulate key developmental processes at the human fetal-maternal interface. Nat Med 2006; 12:1065–1074.
25. Carol M, Lambrechts A, Urbain D, van Vooren JP, Clumeck N, Goldman M, Mascart-Lemone F. Persistent T cell and B cell activities in the duodenal mucosa of AIDS patients. AIDS 1998; 12:1763–1769.
26. Quigley MF, Abel K, Zuber B, Miller CJ, Sandberg JK, Shacklett BL. Perforin expression in the gastrointestinal mucosa is limited to acute simian immunodeficiency virus infection. J Virol 2006; 80:3083–3087.
27. Romagnani C, Juelke K, Falco M, Morandi B, D'Agostino A, Costa R, et al
. CD56brightCD16− killer Ig-like receptor- NK cells display longer telomeres and acquire features of CD56dim NK cells upon activation. J Immunol 2007; 178:4947–4955.