Milk Matters: Soluble Toll-Like Receptor 2 (sTLR2) in Breast Milk Significantly Inhibits HIV-1 Infection and Inflammation

Abstract

The majority of infants who breastfeed from their HIV-positive mothers remain uninfected despite constant and repeated exposure to virus over weeks to years. This phenomenon is not fully understood but has been closely linked to innate factors in breast milk (BM). Most recently we have focused on one such innate factor, soluble Toll-like receptor 2 (sTLR2) for its significant contribution as an inhibitor of inflammation triggered by bacterial and viral antigens. We hypothesized that sTLR2 in BM inhibits immune activation/inflammation and HIV-1 infection. sTLR2 protein profiles were analyzed in HIV-uninfected BM and showed dramatic variability in expression concentration and predominant sTLR2 forms between women. sTLR2 immunodepleted BM, versus mock-depleted BM, incubated with Pam3CSK4 lead to significant increases in IL-8 production in a TLR2-dependant fashion in U937, HEK293-TLR2, and Caco-2. Importantly, TLR2-specific polyclonal and monoclonal antibody addition to BM prior to cell-free R5 HIV-1 addition led to significantly (P<0.01, P<0.001, respectively) increased HIV-1 infection in TZM-bl reporter cells. To confirm these findings, sTLR2-depletion in BM led to significantly (P<0.001) increased HIV-1 infection in TZM-bl cells. Notably, immunodepletion does not allow for the complete removal of sTLR2 from BM, thus functional testing shown here may underestimate the total effect elicited by sTLR2 against HIV-1 and synthetic bacterial ligand. This study provides evidence for the first time that sTLR2 in BM may provide a dual protective role for infants breastfeeding from their HIV-infected mothers by; (1) immunomodulating pro-inflammatory responses to bacterial ligands, and (2) directly inhibiting cell-free HIV-1 infection. Thus, sTLR2 in BM may be critical to infant health and prove beneficial in decreasing vertical HIV-1 transmission to infants.

The oral mucosa immune environment and oral transmission of HIV/SIV

“…Cystatin also shows anti-HIV activity at concentrations observed in saliva (154, 162). In addition, studies have also found a decreased risk of HIV transmission in mothers with elevated breast milk levels of sTLR2 (171), CCL4 (172), erythropoietin (173), IL15 (174) and specific long-chain fatty acids (175), indicating that there may be many additional host factors that can reduce HIV acquisition, following oral exposure.…”

“…Analysis of human milk and saliva has revealed numerous factors that inhibit HIV replication in vitro , including lactoferrin, SLPI, and mucins (see ‘Innate and adaptive oral mucosa immunity to HIV’ section). Other milk constituents, such as sTLR2 (171), CCL4 (172), and IL15 (174), correlate with reduced MTCT in epidemiologic studies. An exhaustive characterization of potential inhibitory factors in breast milk of natural hosts has not been performed, and, to date, there have been no studies of salivary inhibitory factors in natural hosts.…”

Soluble human TLR2 ectodomain binds diacylglycerol from microbial lipopeptides and glycolipids

“…Here, we have demonstrated that hTLR2ED directly binds synthetic, diacyl- (FSL-1 and Pam 2 CSK 4 ) and triacyl-lipopeptides (Pam 3 CSK 4 ), purified LTA from S. aureus, and synthetic phosphatidylinositol mannosides (Pim2 and Pim4), in absence of TLR1 or TLR6. Soluble isoforms of hTLR2ED present in plasma [43], breast milk [43–44], amniotic fluids [45], saliva [46] and expressed in HEK cells [47] can act as a decoys of TLR2 and inhibit NFκB activation by transmembrane TLR2 [48]. The hTLR2ED expressed in insect cells should have the same inhibitory effect as the hTLR2ED from natural sources as the only difference with them would be in the types of glycan structures, which should not be relevant for binding as the glycosylation sites are not located near the binding site of hTLR2ED.…”

“…Therefore, our ligand-binding studies under physiological conditions suggest that ligand competition between secreted isoforms of hTLR2ED and the transmembrane hTLR2 receptor is likely to be responsible for the down-regulation of TLR2 activation. Soluble TLR2 has also been implicated in the pathogenesis of HIV [28, 44]. Thus, we investigated whether hTLR2ED could interact with HIV-1 gp120.…”

Toll like receptors: a new hope on the horizon to treat multiple sclerosis

“…On the contrary, inhibiting MyD88-dependent pathway by TLR 2,4,7,8,9 antagonists can also be efficacious. Another approach that could be used for inhibition of MyD88-dependent pathway is administration of pathogen inhibitors such as anti LPS therapy and soluble TLRs (sTLR) [14]. Furthermore, TLRs tolerance approach can be considered for chronic and prophylactic purposes [15].…”