Assessment of the efficacy of clofazimine alone and in combination with primary agents against Mycobacterium tuberculosis in vitro.

OBJECTIVES: The chemotherapeutic regimens of patients with drug-susceptible (DS)- tuberculosis (TB) comprise four primary anti-TB drugs: rifampicin (RMP), isoniazid (INH), ethambutol (EMB) and pyrazinamide (PZA), administered for six-to-nine months. These drug regimens target the various microbial populations that include actively replicating (AR), slow-replicating (SR) and non-replicating (NR) organisms. Clofazimine (CFZ) has showed benefit in shortening DS-TB treatment in vivo from six to four months when used in combination with this regimen in murine models of experimental infection. However, its antimicrobial efficacy when used in combination with the primary drugs against the various microbial populations of Mycobacterium tuberculosis has not been demonstrated. METHODS: In the current in vitro study, the inhibitory and bactericidal activities of CFZ in combination with the primary anti-TB drugs, RMP, INH and EMB against the AR and SR organisms in planktonic and biofilm-forming cultures, respectively, were evaluated by fractional inhibitory concentration index (FICI) and fractional bactericidal concentration index (FBCI) determinations, using the Loewe Additivity Model. RESULTS: In planktonic cultures, CFZ demonstrated synergistic growth inhibitory activity in combination with RMP and INH individually and collectively. With respect to bactericidal activity, CFZ exhibited synergistic activity only in a two-drug combination with RMP. However, in biofilm-forming cultures, all CFZ-containing anti-TB drug combinations exhibited synergistic inhibitory and bactericidal effects, particularly in combination with RIF and INH. CONCLUSION: Clofazimine exhibited synergistic effects in combination with primary anti-TB drugs against both planktonic and biofilm-forming cultures, showing potential benefit in augmenting treatment outcome when used during standard TB chemotherapy.

Hypermethylation at the CXCR5 gene locus limits trafficking potential of CD8+ T cells into B-cell follicles during HIV-1 infection.

CD8+ T cells play an important role in HIV control. However, in human lymph nodes (LNs), only a small subset of CD8+ T cells express CXCR5, the chemokine receptor required for cell migration into B-cell follicles, which are major sanctuaries for HIV persistence in individuals on therapy. Here, we investigate the impact of HIV infection on follicular CD8+ T cell (fCD8) frequencies, trafficking patterns, and CXCR5 regulation. We show that, although HIV infection results in a marginal increase in fCD8s in LNs, the majority of HIV-specific CD8+ T cells are CXCR5- (non-fCD8s) (P < .003). Mechanistic investigations using Assay for Transposase-Accessible Chromatin using sequencing showed that non-fCD8s have closed chromatin at the CXCR5 transcriptional start site (TSS). DNA bisulfite sequencing identified DNA hypermethylation at the CXCR5 TSS as the most probable cause of closed chromatin. Transcriptional factor footprint analysis revealed enrichment of transforming growth factors (TGFs) at the TSS of fCD8s. In vitro stimulation of non-fCD8s with recombinant TGF-ß resulted in a significant increase in CXCR5 expression (fCD8s). Thus, this study identifies TGF-ß signaling as a viable strategy for increasing fCD8 frequencies in follicular areas of the LN where they are needed to eliminate HIV-infected cells, with implications for HIV cure strategies.