Transcriptomic Evidence of the Immune Response Activation in Individuals With Limb Girdle Muscular Dystrophy Dominant 2 (LGMDD2) Contributes to Resistance to HIV-1 Infection

Abstract

LGMDD2 is a rare form of muscular dystrophy characterized by one of the three heterozygous deletions described within the TNPO3 gene that result in the addition of a 15-amino acid tail in the C-terminus.TNPO3 is involved in the nuclear import of splicing factors and acts as a host cofactor for HIV-1 infection by mechanisms not yet deciphered. Further characterization of the crosstalk between HIV-1 infection and LGMDD2 disease may contribute to a better understanding of both the cellular alterations occurring in LGMDD2 patients and the role of TNPO3 in the HIV-1 cycle. To this regard, transcriptome profiling of PBMCs from LGMDD2 patients carrying the deletion c.2771delA in the TNPO3 gene was compared to healthy controls. A total of 545 differentially expressed genes were detected between LGMDD2 patients and healthy controls, with a high representation of G protein-coupled receptor binding chemokines and metallopeptidases among the most upregulated genes in LGMDD2 patients. Plasma levels of IFN-β and IFN-γ were 4.7- and 2.7-fold higher in LGMDD2 patients, respectively. An increase of 2.3-fold in the expression of the interferon-stimulated gene MxA was observed in activated PBMCs from LGMDD2 patients after ex vivo HIV-1 pseudovirus infection. Thus, the analysis suggests a pro-inflammatory state in LGMDD2 patients also described for other muscular dystrophies, that is characterized by the alteration of IL-17 signaling pathway and the consequent increase of metallopeptidases activity and TNF response. In summary, the increase in interferons and inflammatory mediators suggests an antiviral environment and resistance to HIV-1 infection but that could also impair muscular function in LGMDD2 patients, worsening disease evolution. Biomarkers of disease progression and therapeutic strategies based on these genes and mechanisms should be further investigated for this type of muscular dystrophy.

Keywords: HIV-1; LGMDD2; chemokines; inflammation pathway; transcriptome.

FIGURE 1

Differentially expressed genes with higher FC between LGMDD2 patients and healthy controls. Functional annotation of these genes is shown according to the legend.

FIGURE 2

Enriched pathways of KEGG database associated to differentially expressed genes between LGMDD2 patients and healthy controls. Each node of the network represents an enriched term, the node color represents different clusters, and the node size represent 5 levels of FDR-corrected p-values (p < 0.05, p < 0.01, p < 0.001, p < 0.0001 and p < 0.00001). The FDR-corrected p-value associated to each specific KEGG pathway is showed in the bubble plot.

FIGURE 3

Transcripts per million of reads obtained in the RNA-Seq experiment for differentially expressed genes encoding metallopeptidases. Dashes inside boxes indicated the median value and crosses indicated the arithmetic mean. Box limits indicate the interquartile range (IQR). Whiskers are adjusted to maximal and minimal values if lower than 1.5 times the IQR. Further outliers are indicated as circles (A). RNA-Seq results expressed in transcripts per million reads obtained for LGMDD2 patient according to their severity (B).

FIGURE 4

Inflammatory response in LGMDD2 patients evidenced by altered IL-17 (A) and TNF (B) signaling pathways. Green coloured genes represent overexpressed genes in LGMDD2 patients whereas red coloured genes represent genes downregulated in these individuals.

FIGURE 5

Sensing of PAMPs through TLRs in LGMDD2 patients. Green coloured genes represent overexpressed genes in LGMDD2 patients.

FIGURE 6

Antiviral cytokines profile in plasma samples of healthy controls and LGMDD2 patients. Levels (pg/ml) of antiviral cytokines IFNβ (A) and IFNγ (B) were quantified in plasma of healthy controls and LGMDD2 patients showing a significant increase in both IFN (p-values = 0.0159 and 0.0286, respectively).

FIGURE 7

Type I interferon production. PBMCs isolated from healthy controls and LGMDD2 patients were infected by spinoculation with two different HIV-1 strains: VSV-luc and NL4-3_renilla. The production of type I IFN was measured 48 h post infection by quantifying MxA expression by flow cytometry.