Here we report on predominant forms of sTLR2 in breast milk (BM) that vary between women and show that they have direct effects on inflammatory responses and HIV-1 infection. Results confirm previous findings that indicate sTLR2 is critical in suppressing inflammatory responses to bacterial PAMPs 
, and extend to show direct inhibition of cell-free R5 HIV-1 infection. Thus, sTLR2 may be an important innate factor that protects infants breastfeeding from HIV positive mothers by (1) directly inhibiting cell-free R5 HIV-1 infection, and (2) inhibiting immune activation, namely inflammation of the nursing infant’s gut.
To our knowledge, this is the first time that the major 38 kDa and 26 kDa sTLR2 polypeptides have been reported as predominant sTLR2 forms in BM, although similar-sized sTLR2 forms have been observed in both amniotic fluid, saliva 
, and cervicovaginal fluid (Henrick et al., unpublished data). Variation in predominant sTLR2 polypeptides between different women’s BM samples may be the result of racial, ethnic or genetic variability, which may explain the differences in predominant sTLR2 forms identified between our cohort and the one described by LeBouder et al
(2003), despite the similarity in sample collection, times postpartum, processing, and Western blot analysis, including antibodies used. Notably, we provide data that multiple TLR2-specific mAbs were unable to detect commercially available human rsTLR2 indicating important structural, and/or conformational differences that were further associated with altered immune functionality in vitro
. Indeed, rsTLR2 is produced in mouse myeloma cell lines, which may alter glycosylation patterns as opposed to native proteins, and may prove pivotal given the undoubted importance of N-linked glycosylation in proper sTLR2 synthesis, secretion, and function 
. These data indicate that caution should be used when interpreting data in which commercially available rsTLR2 is used as a replacement for natural sTLR2 forms. Further, interpretation of our data leads us to believe that intermolecular non-covalent interactions between the predominant sTLR2 polypeptide 38 kDa and 26 kDa are responsible for sTLR2 protein complexes that function in a coherent manner to inhibit bacterial PAMP-induced production of pro-inflammatory cytokine production and R5 HIV-1 infection.
Further, experiments into the regulation of major sTLR2 polypeptides in BM may be important in fully appreciating the intricacies of intestinal microbiome development in infants. Our observations indicated that multiple cells of the mammary gland and BM contribute to the generation of 26 kDa sTLR2, however the source of the 38 kDa form remains elusive. Data indicate that 38 kDa sTLR2 production is down-regulated over time postpartum and is susceptible to degradation at room temperature. These observations may correlate with the infant’s maturing immune system 
, and assist in the development of tolerance as well as proper IEC epithelial barrier function 
The function of BM sTLR2 has been shown to be immunomodulatory by providing direct attenuation of signaling through membrane TLR2 while still allowing for effective elimination of the pathogen in a mouse model 
. Here, we demonstrated that sTLR2 in BM has dual utility by providing both anti-inflammatory and anti-viral functions: (1) sTLR2 depleted BM led to significantly increased IL-8 pro-inflammatory cytokine production in U937, HEK293-TLR2 and human IECs exposed to PAM3
; (2) Inhibition of sTLR2 through the addition of TLR2-specific antibodies as well as sTLR2-depleted BM led to significantly increased HIV-1 infection in TZM-bl reporter assays. To the best of our knowledge, this is the first study to demonstrate that sTLR2 in BM can directly inhibit cell-free R5 HIV-1 infection. However, previous studies have demonstrated a strong correlation between decreased sTLR2 concentrations and HIV-1 progression 
, and high levels of TLR2 expression on monocytes taken from HIV-1 infected patients 
. Indeed, there are numerous publications indicating that many viruses including measles, VSV, CMV, hepatitis C and herpes virus activate TLR2 through viral glycoproteins leading to the production of proinflammatory cytokines 
. Further, we previously showed significantly increased TLR2 expression in peripheral blood mononuclear cells of Kenyan commercial sex workers with AIDS, which is reduced with HAART treatment 
. Our data is timely given a recent publication confirming increased levels of HIV-1 RNA in BM positively correlate with vertical transmission 
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. Likewise, this technique has experimental limitations that do not preclude the possibility that an unknown binding partner of sTLR2 may also contribute for the reduction of pro-inflammatory cytokine production in response to bacterial ligands and HIV-1 inhibition shown here. Future research is warranted to determine the exact mechanism of sTLR2 functionality including the involvement of cofactor CD14 in the direct binding and inhibition of HIV, as the necessity of the cofactor differs between viral agonists 
In conclusion, the current 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. We confirm that BM sTLR2 complexes inhibit production of pro-inflammatory cytokines during bacterial ligand exposure, and extend the functional role of BM sTLR2 complexes to include inhibition of cell-free R5 HIV-1 infection. As discussed previously, immune activation has marked effects on HIV-1 acquisition (reviewed in 
), therefore, data shown here may prove critical in understanding vertical HIV-1 transmission through BM, and indicates sTLR2 may be an important factor in a complex milieu of immunomodulatory and anti-viral factors that help protect the majority of infants from becoming infected despite repeated and prolonged exposure to HIV-1 infected BM.