Sexually Dimorphic Role of Toll-like Receptor 4 (TLR4) in High Molecular Weight Hyaluronan (HMWH)-induced Anti-hyperalgesia

Animals

Experiments were performed on 220–400 g female and male Sprague-Dawley rats (Charles River Laboratories, Hollister, CA, USA). Experimental animals were housed three per cage, under a 12-hour light/dark cycle, in a temperature- and humidity-controlled animal care facility at the University of California, San Francisco. Food and water were available ad libitum. Experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of California, San Francisco, and adhered to the National Institutes of Health guidelines for the care and use of laboratory animals.

Measuring nociceptive threshold

Mechanical nociceptive threshold was quantified using an Ugo Basile Analgesymeter (Stoelting, Wood Dale, IL, USA). This device applies a linearly increasing mechanical force to the dorsum of a rat’s hindpaw ^{38, 42, 43}. Rats were placed in acrylic cylindrical restrainers, with lateral ports to allow access to the leg and hind paw, as described previously ⁵. Rats were acclimatized to the restrainers over a 3-day period to habituate them to the testing procedure. Mechanical nociceptive threshold is defined as the force in grams at which a rat withdrew its paw. Baseline threshold is defined as the mean of three readings taken before injection of test agents. Each experiment was performed on a different group of rats. Data are presented as mean ± SEM percentage change from baseline nociceptive threshold.

Drugs

The following drugs were used in the present experiments: high molecular weight hyaluronan (HMWH) [hyaluronic acid sodium salt from Streptococcus pyogenes], from Calbiochem (San Diego, CA, USA); prostaglandin E₂ (PGE₂), ST2825 (a myeloid differentiation factor 88 [MyD88] inhibitor), BX-795 (an IRF3 inhibitor), dihydrotestosterone and 17β-estradiol all from Sigma-Aldrich (Saint Louis, MO, USA); and NBP2–26245 (a TLR4 receptor antagonist) from GenScript (Piscataway, NJ, USA).

PGE₂ was dissolved in absolute ethanol to a concentration of 1 μg/μL, and immediately before experiments further diluted in 0.9% saline to the concentration used in each experiment. The ethanol concentration of the final PGE₂ solution was ~2%, a concentration previously shown to not affect mechanical nociceptive threshold ²². HMWH, was dissolved in distilled water to a concentration of 1 μg/μL, and further diluted in 0.9% saline to the concentration used in each experiment. Aliquots containing 1 μg/μL of BX-795 and ST2825, dissolved in 100% dimethyl sulfoxide (DMSO), were diluted in 0.9% NaCl containing 10% DMSO to a concentration of 0.2 μg/μL.

All intradermal administered drugs were in a volume of 5 μL (when injected alone) or 3 μL each (when two or more drugs were co-injected), on the dorsum of the hind paw, using a 30-gauge hypodermic needle attached to a 50 μL Hamilton syringe by a segment of PE-10 polyethylene tubing (Becton Dickinson, Franklin Lakes, NJ, USA). The administration of BX-795 and ST2825 was preceded by a hypotonic shock (1 μL of distilled water, separated by a bubble of air to avoid mixing in the same syringe), distilled water produces hypo-osmotic shock, allowing drugs to penetrate the cell plasma membrane, to get these two reagents inside the submicron diameter nociceptor nerve terminal ^{11, 14}.

Oligodeoxynucleotides (ODN) antisense to CD44 and toll-like receptor 4 (TLR4) mRNA

The role of CD44 and TLR4 in the anti-nociceptive effects of HMWH, were assessed in male and female rats treated intrathecally with ODN antisense to each receptor’s mRNA. ODN antisense was directed against a unique region of the rat mRNA sequence for each receptor. ODN antisense sequences were:

• CD44 ODN antisense: 5’-GAA AAG GGT CGC GGG GG-3’ (GenBank accession number NM_012924.2)

• TLR4 ODN antisense: 5’-AGG AAG TGA GAG TGC CAA CC-3’ (GenBank accession number NM_019178.1)

ODN mismatch sequences correspond to the antisense sequence with mismatched bases (denoted by bold letters). ODN mismatch sequences:

• CD44 ODN mismatch: 5’-CCC CCG CGA CCC TTT TC-3’

• TLR4 ODN mismatch: 5’-ACG ATG CGA GAG AGT CAC CG-3’

ODNs were synthesized by Life Technologies (Carlsbad, CA, USA), and have been previously shown to produce a decrease in CD44 and TLR4 protein ^{5, 9}. Before use, ODNs were reconstituted in nuclease-free 0.9% NaCl and then administered intrathecally, for 3 consecutive days ⁵. As described previously ², rats were anesthetized with isoflurane (2.5% in O₂) and ODN (120 μg in 20 μL) injected intrathecally using a 300 μl syringe attached to a 29-gauge hypodermic needle that was inserted into the subarachnoid space between the L4 and L5 vertebrae. The intrathecal site of injection was confirmed by a sudden flick of the rat’s tail, a reflex that is evoked by subarachnoid space access and bolus intrathecal injection ³³. Animals regained consciousness approximately 2 minutes after the injection and termination of anesthesia. The use of ODN antisense administered intrathecally to attenuate the expression of proteins essential for nociceptor sensitization is well supported by previous studies, by others ^{35, 37, 39–41} as well as our group ^{4–6, 8, 21–23, 36}.

Gonadectomy

Gonadectomy, was performed on 22–25 day old male and female rats (i.e., before sexual maturation); gonadectomized rats were used for experiments 3 weeks later (i.e., as adults) ²⁷. For this surgery, animals were anesthetized with isoflurane (3% in oxygen) and received preoperative meloxicam (~5 mg/kg, s.c.), and bupivacaine (~0.1 mg/kg s.c. infiltrated at incision site) for pain control.

Statistical analysis

The dependent variable in behavioral experiments was percentage change from baseline mechanical paw-withdrawal threshold. We used 186 male and 96 female rats. In each experiment only one hind paw per rat was used. The behavioral experiments were performed with the experimenter blinded to experimental group. One-way ANOVA followed by Bonferroni’s post hoc comparisons test, or Student’s t-test, was used to analyze data, as appropriate, described in each figure legend. Prism 8.0 (GraphPad Software) was used for the graphics and to perform statistical analyses; P<0.05 was considered statistically significant. Data are presented as mean ± SEM.

TLR4 dependence of HMWH-induced anti-hyperalgesia

To test the hypothesis that HMWH-induced anti-hyperalgesia is TLR4 dependent in male rats, male (Fig. 1A), female (Fig. 1B) and ovariectomized female (Fig. 1C) rats received an intradermal injection of PGE₂ (100 ng, i.d.) followed by a TLR4 antagonist (NBP2–26245, 1 μg, i.d.), all injected at the site of nociceptive testing, and then 5 min later HMWH (1 μg, i.d.) was injected. Male rats receiving the TLR4 antagonist showed attenuation of HMWH-induced anti PGE₂ hyperalgesia. While in female rats, the TLR4 antagonist did not attenuate HMWH-induced anti-hyperalgesia, in ovariectomized female rats it significantly attenuated HMWH-induced anti-hyperalgesia.

TLR4 signaling pathway mediating HMWH-induced anti-hyperalgesia

Since HA can signal via TLR4 ³², we screened for a role of TLR4 second messenger pathways in HMWH-induced anti-hyperalgesia. TLR4 has two well-described second messenger signaling pathways, MyD88-dependent and MyD88-independent. To test the hypothesis that HMWH signals through the MyD88-dependent pathway, to induce anti-hyperalgesia, male rats were treated intradermally with PGE₂ (100 ng, i.d.), followed 5 min later by a MyD88 inhibitor (ST2825, 1 μg, i.d.) and then, 5 min later by HMWH (1 μg, i.d.); all injections were performed at the site of nociceptive testing, on the dorsum of the hind paw. Male rats treated with the MyD88 inhibitor showed attenuation of HMWH-induced anti-hyperalgesia (Fig. 2).

The MyD88-independent TLR4 pathway involves TIR-domain-containing adaptor inducing interferon-β (TRIF) and the recruitment of the TRIF-related Adaptor Molecule (TRAM). To determine if the MyD88-independent pathway also mediates the TLR4-dependent contribution to HMWH-induced anti-hyperalgesia, we administered PGE₂ (100 ng, i.d.), followed 5 min later by an inhibitor of IRF3, a component of the MyD88-independent signaling pathway (BX-795, 1 μg, i.d.), and then 5 min later HMWH (1 μg, i.d.). Male rats treated with the IRF3 inhibitor did not show attenuation of HMWH-induced anti-hyperalgesia (Fig. 3).

Role of sex hormones in TLR4 dependence

We compared the effect of ODN antisense to TLR4 mRNA on HMWH anti-hyperalgesia in gonad-intact and gonadectomized male and female rats, with or without same sex hormone replacement (Fig. 4). In TLR4 antisense-treated rats, we administered PGE₂ (100 ng, i.d.), followed 10 min later by HMWH (1 μg, i.d.). TLR4 ODN antisense attenuated HMWH-induced anti-hyperalgesia in gonad-intact male (Fig. 4A), but not in gonad-intact female rats (Fig. 4B). Orchiectomized male rats treated with TLR4 ODN antisense still demonstrated attenuation of HMWH-induced anti-hyperalgesia (Fig. 4A), while ovariectomized female rats now demonstrated attenuation of HMWH-induced anti-hyperalgesia (Fig. 4B). Next, the group of ovariectomized female rats were replaced with 17β-estradiol, and the group of orchiectomized male were treated with dihydrotestosterone. In orchiectomized male rats treated with dihydrotestosterone, TLR4 ODN antisense attenuated HMWH anti-hyperalgesia (Fig. 4A). And, in hormone-replaced ovariectomized female rats, HMWH-induced anti-hyperalgesia was again not attenuated by ODN antisense to TLR4 mRNA (Fig. 4B).

Effect of combining TLR4 and CD44 antisense

Since both TLR4 and CD44 antisense partially attenuate HMWH anti-hyperalgesia, in male rats, we administered the combination of TLR4 plus CD44 ODN antisense. Male rats that received both TLR4 and CD44 ODN antisense showed almost complete reversal of the anti-hyperalgesia induced by HMWH (Fig. 5).