Quantitation of gene expression in formaldehyde-fixed and fluorescence-activated sorted cells.

Fluorescence-activated cell sorting (FACS) is a sensitive and valuable technique to characterize cellular subpopulations and great advances have been made using this approach. Cells are often fixed with formaldehyde prior to the sorting process to preserve cell morphology and maintain the expression of surface molecules, as well as to ensure safety in the sorting of infected cells. It is widely recognized that formaldehyde fixation alters RNA and DNA structure and integrity, thus analyzing gene expression in these cells has been difficult. We therefore examined the effects of formaldehyde fixation on the stability and quantitation of nucleic acids in cell lines, primary leukocytes and also cells isolated from SIV-infected pigtailed macaques. We developed a method to extract RNA from fixed cells that yielded the same amount of RNA as our common method of RNA isolation from fresh cells. Quantitation of RNA by RT-qPCR in fixed cells was not always comparable with that in unfixed cells. In comparison, when RNA was measured by the probe-based NanoString system, there was no significant difference in RNA quantitation. In addition, we demonstrated that quantitation of proviral DNA in fixed cells by qPCR is comparable to that in unfixed cells when normalized by a single-copy cellular gene. These results provide a systematic procedure to quantitate gene expression in cells that have been fixed with formaldehyde and sorted by FACS.

Detection of microbial translocation in HIV and SIV infection using the Limulus amebocyte lysate assay is masked by serum and plasma.

OBJECTIVE: Microbial translocation (MT) is thought to be a major contributor to the pathogenesis of HIV-related immune activation, and circulating lipopolysaccharide (LPS) from gram-negative bacteria is the principle measurement of this process. However, related research has been impeded by inconsistent LPS test results. METHODS: Specimens were obtained from HIV-infected adults enrolled in the PEARLS study (ACTG A5175) and HIV-HCV co-infected participants enrolled in a study of liver disease staging using MRI elastography. Pig-tailed macaque specimens were obtained from SIV-infected and -uninfected animals. Samples were tested for LPS using the LAL assay with diazo-coupling modifications to improve sensitive detection. RESULTS: When exogenous LPS was added to macaque plasma, >25% inhibition of LPS detection was found in 10/10 (100%) samples at 20% plasma concentration compared to control; in contrast 5/10 (50%) samples at 2% plasma concentration (p = 0.07) and 0/10 (0%) at 0.1% plasma concentration (p = 0.004) showed >25% inhibition of LPS detection. Similarly, when LPS was added to human serum, >25% inhibition of LPS detection was found in 5/12 (42%) of samples at 2% serum concentration compared to control, while 0/12 (0%) of samples in 0.1% serum showed >25% inhibition of LPS detection (p = 0.07). Likewise, LPS detection in human sera without exogenous LPS was improved by dilution: LPS was detected in 2/12 (17%) human samples in 2% serum, ranging from 3,436-4,736 pg/mL, compared to 9/12 (75%) samples in 0.1% serum, ranging from 123 pg/mL -60,131 pg/mL (p = 0.016). In a separate validation cohort of HIV-HCV co-infected participants sampled at two different times on the same day, LPS measured in 0.2% plasma and with diazo-coupling was closely correlated between the first and second samples (R = 0.66, p<0.05). CONCLUSIONS: Undiluted serum and plasma mask LPS detection. The extent of MT may be substantially underestimated.

Expansion of a subset of CD14highCD16negCCR2low/neg monocytes functionally similar to myeloid-derived suppressor cells during SIV and HIV infection.

Monocytes have been categorized in three main subpopulations based on CD14 and CD16 surface expression. Classical monocytes express the CD14(++)CD16(-)CCR2(+) phenotype and migrate to inflammatory sites by quickly responding to CCL2 signaling. Here, we identified and characterized the expansion of a novel monocyte subset during HIV and SIV infection, which were undistinguishable from classical monocytes, based on CD14 and CD16 expression, but expressed significantly lower surface CCR2. Transcriptome analysis of sorted cells demonstrated that the CCR2(low/neg) cells are a distinct subpopulation and express lower levels of inflammatory cytokines and activation markers than their CCR2(high) counterparts. They exhibited impaired phagocytosis and greatly diminished chemotaxis in response to CCL2 and CCL7. In addition, these monocytes are refractory to SIV infection and suppress CD8(+) T cell proliferation in vitro. These cells express higher levels of STAT3 and NOS2, suggesting a phenotype similar to monocytic myeloid-derived cells, which suppress expansion of CD8(+) T cells in vivo. They may reflect an antiproliferative response against the extreme immune activation observed during HIV and SIV infections. In addition, they may suppress antiviral responses and thus, have a role in AIDS pathogenesis. Antiretroviral therapy in infected macaque and human subjects caused this population to decline, suggesting that this atypical phenotype is linked to viral replication.