Modified High-Molecular-Weight Hyaluronan Promotes Allergen-Specific Immune Tolerance

Abstract

The extracellular matrix in asthmatic lungs contains abundant low-molecular-weight hyaluronan, and this is known to promote antigen presentation and allergic responses. Conversely, high-molecular-weight hyaluronan (HMW-HA), typical of uninflamed tissues, is known to suppress inflammation. We investigated whether HMW-HA can be adapted to promote tolerance to airway allergens. HMW-HA was thiolated to prevent its catabolism and was tethered to allergens via thiol linkages. This platform, which we call "XHA," delivers antigenic payloads in the context of antiinflammatory costimulation. Allergen/XHA was administered intranasally to mice that had been sensitized previously to these allergens. XHA prevents allergic airway inflammation in mice sensitized previously to either ovalbumin or cockroach proteins. Allergen/XHA treatment reduced inflammatory cell counts, airway hyperresponsiveness, allergen-specific IgE, and T helper type 2 cell cytokine production in comparison with allergen alone. These effects were allergen specific and IL-10 dependent. They were durable for weeks after the last challenge, providing a substantial advantage over the current desensitization protocols. Mechanistically, XHA promoted CD44-dependent inhibition of nuclear factor-κB signaling, diminished dendritic cell maturation, and reduced the induction of allergen-specific CD4 T-helper responses. XHA and other potential strategies that target CD44 are promising alternatives for the treatment of asthma and allergic sinusitis.

Keywords: T cell; allergy; dendritic cell; hyaluronan; tolerance.

Figure 1.

Chemically modified high-molecular-weight hyaluronan (HMW-HA) (XHA) coupled with ovalbumin (OVA) (OVA/XHA) prevents allergic airway inflammation in mice previously sensitized to OVA. (A) Agarose gel electrophoresis of unmodified 1.56 × 106 443 Da HMW-HA and thiolated, 500 kD HMW-HA treated with increasing concentrations of hyaluronidase (HA’ase). A size standard (SS) ladder of 400–100 kD hyaluronan (HA) is also shown. (B) Agarose gel electrophoresis of unmodified 1.56 × 106 445 Da HMW-HA thiolated, 500 kD HMW-HA ± polyethylene (glycol) diacrylate (PEGDA) crosslinker (XHA) and controls. XHA was crosslinked overnight. All reagents were purchased commercially, and the aforementioned HA size characteristics are per the manufacturers’ information. Data in A and B are each representative of three experiments. (C) Schematic of the OVA-induced allergic airway inflammation protocol. (D) Total cell counts and (E) leukocyte subsets in bronchoalveolar lavage (BAL) fluid of mice rechallenged with OVA/XHA. Data in D and E are representative of eight independent experiments. Total cell counts (F) and eosinophils (G) in BAL fluid of mice treated with OVA, OVA/XHA, low-molecular-weight hyaluronan (LMW-HA), or alginate (ALG). *P < 0.05 by Student’s t test. SE is shown. Macs, macrophages.

Figure 2.

OVA/XHA prevents physiologic, histologic, and immunologic manifestations of allergic airway inflammation in mice sensitized previously to OVA. Average (A) and maximal (B) lung resistance in response to methacholine challenge. (C) Total BAL fluid (BALF) cell counts for the same mice as in A and B. (D) Pulmonary draining lymph node (PDLN) cell numbers for mice sensitized with OVA and then subsequently challenged with OVA or OVA/XHA. (E) OVA-specific IgE levels in BALF isolated from sensitized mice challenged with OVA or OVA/XHA. Data are for BAL pooled for five mice per group and measured in triplicate. Representative hematoxylin and eosin–stained lung sections for mice treated with (F) PBS, (G) OVA, or (H) OVA/XHA. Cytokine mRNA expression in pulmonary tissue of mice treated with OVA or OVA/XHA, specifically (I) IL-4, (J) IL-13, (K) IL-17, and (L) IFN-γ, all normalized to 18S expression. Tissues from five animals were pooled for this experiment and measured in triplicate. *P < 0.05 by a Student’s t test. SE is shown.

Figure 3.

Antiinflammatory effects of XHA on allergic airway inflammation are durable for 2 weeks. (A) Schematic of the protocol used to evaluate the durability of OVA/XHA-mediated tolerization. (B) BALF cell counts in response to challenge with OVA alone, in mice that received OVA/XHA 12–16 days earlier. Data are representative of three independent experiments. *P < 0.05 by Student’s t test. SE is shown. ns, not significant.

Figure 4.

XHA treatment inhibits proliferative responses to allergen but not to polyclonal stimulus. (A–C) PDLN lymphocytes and (D–F) splenocytes were isolated from animals sensitized to OVA and then challenged with PBS, OVA, or OVA/XHA. Cell counts in (A) PDLN and (D) spleens from mice treated with PBS or OVA or OVA/XHA are shown. Lymphocytes and splenocytes were then stimulated ex vivo with either (B and E) OVA or (C and F) anti-CD3 at the concentrations indicated for 72 hours. Tritiated thymidine was added for the final 24 hours. Data are representative of two independent experiments. In a separate experiment, PDLN lymphocytes and splenocytes were stained directly ex vivo for (G) I-A/I-E, (H) CD80, (I) CD86, and (J) CD205. MFIs were calculated relative to antibody-appropriate isotype controls. *P < 0.05; **P < 0.01. Data are representative of two experiments. CPM, counts per minute; MFI, median fluorescence intensity; PLN, pulmonary lymph node.

Figure 5.

OVA/XHA effects are IL-10 dependent. Mice sensitized previously to OVA were administered intranasal OVA or OVA/XHA. Cells were isolated from total BAL fluid via centrifugation (4 × 1 ml flushes of the lung with PBS) and were activated in vitro with anti-CD3 for 48 hours. (A) IL-10 levels in cell culture supernatants as measured by ELISA. (B) BAL fluid counts for the same animals as in A. The same protocol was repeated in IL-10^−/− mice, and total cell counts were measured in (C) BAL fluid and (D) PLN. *P < 0.05. LN, lymph node.

Figure 6.

XHA prevents bone marrow–derived dendritic cell (BMDC) maturation in response to LPS. BMDC were treated with LPS for 24 hours in the setting of a 1% coating of XHA or soluble LMW-HA. Representative histograms showing the change in cell surface markers including (A) I-A/I-E, (B) CD14, (C) CD62L (D) CD80, and (E) CD86 after treatment with XHA and controls. (F) Fold change in MFI for the same markers as in A–E. This figure includes data from three independent experiments. Representative appearance of BMDC cultured in the setting of (G) PBS, (H) LPS, (I) LPS and XHA, or (J) LPS and LMW-HA. (K) Proliferation in response to OVA, as measured by carboxyfluorescein succinimidyl ester (CFSE) dye dilution, for CD4⁺T491 cells isolated from animals immunized previously with OVA and cultured for 72 hours with BMDC cultured previously for 24 hours with LPS ± XHA or LMW-HA. Western blot for the p65 subunit of nuclear factor-κB (NF-κB) for (L) whole cell lysates or (M) the corresponding cytosolic (C) and nuclear (N) fractions of dendritic cells, treated with PBS or LPS ± XHA. (N) Ratios of C to N NF-κB in lysates for three independent experiments. *P < 0.05 by Student’s t test. SE is shown.

Figure 7.

XHA-mediated inhibition of allergic airway inflammation is CD44 dependent. (A) BAL cell counts for CD44^−/− mice after rechallenge with OVA or OVA/XHA. (B) BAL cell counts for wild-type (CD44^+/+) mice treated with OVA, OVA/XHA, or anti-CD44 antibody (Ab). (C) BAL cell counts for mice treated as in B but now for different leukocyte subsets. *P < 0.05. Data in A–C are representative of three experiments. (D) Proliferation in response to OVA, as measured by CFSE dye dilution, for CD4⁺T cells isolated from animals immunized with OVA and cultured for 72 hours with BMDC cultured previously for 24 hours with LPS ± XHA or anti-CD44 Ab. Western blots for the p65 subunit of NF-κB generated using (E) whole cell lysates or (F) the corresponding C and N fractions of dendritic cells treated with PBS or LPS ± aCD44 Ab or an isotype-matched control IgG. (G) Ratios of C to N NF-κB in lysates of dendritic cells treated as in F, now for three independent experiments. (H–J) Similar data as shown in Figures 6L–6N only now generated using cells from CD44^−/− mice. *P < 0.05; **P < 0.01 by Student’s t test. SE is shown.