Basophil-derived IL-4 promotes cutaneous Staphylococcus aureus infection

Superficial cutaneous Staphylococcus aureus (S. aureus) infection in humans can lead to soft tissue infection, an important cause of morbidity and mortality. IL-17A production by skin TCRγδ+ cells in response to IL-1 and IL-23 produced by epithelial and immune cells is important for restraining S. aureus skin infection. How S. aureus evades this cutaneous innate immune response to establish infection is not clear. Here we show that mechanical injury of mouse skin by tape stripping predisposed mice to superficial skin infection with S. aureus. Topical application of S. aureus to tape-stripped skin caused cutaneous influx of basophils and increased Il4 expression. This basophil-derived IL-4 inhibited cutaneous IL-17A production by TCRγδ+ cells and promoted S. aureus infection of tape-stripped skin. We demonstrate that IL-4 acted on multiple checkpoints that suppress the cutaneous IL-17A response. It reduced Il1 and Il23 expression by keratinocytes, inhibited IL-1+IL-23–driven IL-17A production by TCRγδ+ cells, and impaired IL-17A–driven induction of neutrophil-attracting chemokines by keratinocytes. IL-4 receptor blockade is shown to promote Il17a expression and enhance bacterial clearance in tape-stripped mouse skin exposed to S. aureus, suggesting that it could serve as a therapeutic approach to prevent skin and soft tissue infection.


Introduction
Staphylococcus aureus (S. aureus) is the most common causative agent of bacterial skin and soft tissue infections (SSTIs) (1)(2)(3). Impetigo, a superficial skin infection that commonly affects children, can lead to SSTIs. S. aureus commonly infects the skin of healthy individuals following trauma, burns, or surgical interventions, as well as the skin of patients with chronic diseases characterized by itching, such as atopic dermatitis (AD) and diabetes, suggesting that skin barrier disruption predisposes to cutaneous S. aureus infection (3)(4)(5)(6).
Basophils account for less than 1% of peripheral blood leukocytes. Basophils produce IL-4 and IL-13 and have been identified as important players in type 2 immune responses against allergens and parasitic infection (23). IL-4 has been shown to inhibit IL-17A production via STAT6 inhibition to the Il17a promoter (24). Basophils may promote Th17 responses by secreting IL-6 (25-28). Moreover, basophil-derived Superficial cutaneous Staphylococcus aureus (S. aureus) infection in humans can lead to soft tissue infection, an important cause of morbidity and mortality. IL-17A production by skin TCRγδ + cells in response to IL-1 and IL-23 produced by epithelial and immune cells is important for restraining S. aureus skin infection. How S. aureus evades this cutaneous innate immune response to establish infection is not clear. Here we show that mechanical injury of mouse skin by tape stripping predisposed mice to superficial skin infection with S. aureus. Topical application of S. aureus to tape-stripped skin caused cutaneous influx of basophils and increased Il4 expression. This basophil-derived IL-4 inhibited cutaneous IL-17A production by TCRγδ + cells and promoted S. aureus infection of tape-stripped skin. We demonstrate that IL-4 acted on multiple checkpoints that suppress the cutaneous IL-17A response. It reduced Il1 and Il23 expression by keratinocytes, inhibited IL-1+IL-23-driven IL-17A production by TCRγδ + cells, and impaired IL-17A-driven induction of neutrophil-attracting chemokines by keratinocytes. IL-4 receptor blockade is shown to promote Il17a expression and enhance bacterial clearance in tape-stripped mouse skin exposed to S. aureus, suggesting that it could serve as a therapeutic approach to prevent skin and soft tissue infection.
The mechanisms by which S. aureus circumvents the innate immune response to establish infection in injured skin are not well understood. We show that tape stripping, which only disrupts the upper layers of the epidermis, rendered mouse skin susceptible to S. aureus infection and that IL-4 derived from basophils recruited into S. aureus-exposed, tape-stripped skin inhibited cutaneous IL-17A production and promoted S. aureus skin infection. We further demonstrate that IL-4 inhibited Il1b and Il23 expression in keratinocytes, acted on TCRγδ + cells to inhibit their IL-17A production in response to IL-1β and IL-23, and blocked IL-17A-driven induction of neutrophil-attracting chemokines in keratinocytes. Furthermore, we show that IL-4 signaling blockade reversed the susceptibility of tape-stripped skin to S. aureus infection.

Results
Mechanical skin injury promotes superficial cutaneous infection by S. aureus. We examined the clearance of topically applied S. aureus from shaved skin without or with tape stripping. Tape stripping resulted in a significant increase in transepidermal water loss (TEWL) that persisted for 72 hours ( Figure 1A), demonstrating effective disruption of the skin barrier. A total of 1 × 10 8 colony-forming units (CFU) of community-acquired methicillin-resistant S. aureus (MRSA; parental strain USA300 SF8300) labeled with the fluorescent dye PSVue 794 were applied topically. The SF8300 MRSA strain used in this study was the most prevalent S. aureus strain causing infections in the United States in the early 2000s, is one of the better characterized MRSA strains, and is the one most commonly used in laboratory studies (30)(31)(32). Persistence of S. aureus was determined at 0, 24, 48, and 72 hours by in vivo fluorescence imaging, as well as by measuring the numbers of CFU in skin homogenates. S. aureus was virtually completely cleared from shaved skin 24 hours after its application, as evidenced by the extinction of PSVue 794 fluorescence ( Figure 1B) and by the recovery of negligible numbers of CFU at 24 hours and none at 48 and 72 hours ( Figure 1C). In contrast, the decay in PSVue 794 fluorescence was significantly slower after application of S. aureus to tape-stripped skin, persisting throughout 48 hours ( Figure 1B). Moreover, the numbers of CFU recovered at 24 hours were more than half those applied on day 0, and viable S. aureus bacteria were consistently recovered at 48 and 72 hours ( Figure 1C).
Histologic examination of skin sections taken on day 3 revealed that tape stripping caused epidermal hyperplasia that was further increased by application of S. aureus ( Figure 1D). Importantly, Gram staining revealed the presence of Gram-positive bacteria in the epidermis of tape-stripped, but not shaved, skin exposed to S. aureus ( Figure 1E). Immunofluorescence examination on day 3 following topical application of GFP-expressing, community-acquired MRSA strain USA300 LAC revealed the presence of GFP + bacteria in the epidermis of tape-stripped skin but not shaved skin ( Figure 1F). Taken together, these results indicate that mechanical injury of skin promotes superficial cutaneous infection with S. aureus. IL-17A from TCRγδ + cells restrains S. aureus infection of mechanically injured skin. IL-17A is important for the clearance of intradermally injected S. aureus (8,14). We examined the IL-17A response to superficial skin infection with S. aureus. Il17a expression was not detectable in shaved mouse skin but was minimally induced by tape stripping (Figure 2A). S. aureus application modestly upregulated Il17a expression in shaved skin (Figure 2A). In contrast, it caused a drastic increase in Il17a expression in tape-stripped skin (Figure 2A). S. aureus application to shaved skin caused minimal or no increase in the expression of the neutrophil-attracting chemokines Cxcl1, Cxcl2, and Cxcl3; modest neutrophil infiltration; and no detectable increase in the expression of the AMP-encoding genes cathelicidin AMP (Camp) and defensin beta 14 (Defb14) (Figure 2 To investigate whether IL-17A restrains superficial skin infection by S. aureus, we examined Il17a -/mice. Skin fluorescence 24 hours following application of PSVue 794-labeled S. aureus to tape-stripped skin was significantly higher in Il17a -/mice compared with WT controls ( Figure 2E). Moreover, the numbers of S. aureus CFU recovered from skin homogenates on day 3 were significantly higher in Il17a -/mice compared with WT controls ( Figure 2F). As expected, cutaneous expression of Cxcl1, Cxcl2, Cxcl3; neutrophil infiltration; and expression of Camp, Defb4, and Defb14 after S. aureus application to tape-stripped skin were all significantly reduced in Il17a -/mice compared with WT controls (Figure 2, G-I, and Supplemental Figure 1C). TCRγδ + cells, CD4 + TCRαβ + Th17 cells, and ILC3s produce IL-17A (7). Tcrd -/mice, which lack TCRγδ + cells, but have normal numbers of TCRαβ + cells and ILC3s, failed to upregulate cutaneous Il17a expression and had decreased neutrophil influx and higher loads of S. aureus following S. aureus application to tape-stripped skin ( Figure 2, J-L). In addition, intracellular flow cytometry demonstrated that TCRγδ + cells but not CD4 + T cells or ILC3s isolated from S. aureus-infected skin were the main source of IL-17A produced after PMA and ionomycin stimulation (Supplemental Figure 2). These results indicate the importance of IL-17A produced by TCRγδ + cells in clearing S. aureus infection from mechanically injured skin. IL-1 and IL-23 promote cutaneous Il17a expression in mechanically injured skin infected by S. aureus. The cytokines IL-1 and IL-23 drive IL-17A production by TCRγδ + cells (16). Expression of Il1a, Il1b, and Il23p19 in tape-stripped skin markedly increased following application of S. aureus ( Figure 3A). Keratinocytes express predominantly Il1a while both keratinocytes and myeloid cells express Il1b and Il23p19 (33)(34)(35)(36)(37). Il1a expression in tape-stripped skin exposed to S. aureus was confined predominantly to the epidermis. In contrast, Il1b was expressed predominantly in the dermis. Il23p19 was expressed in both layers but significantly more in epidermis than dermis ( Figure 3B). Upregulation of Il23p19 expression in tape-stripped skin exposed to S. aureus was virtually abolished in Il1r1 -/mice ( Figure 3C). Moreover, recombinant IL-1α (rIL-1α) induced Il23p19 expression in epidermal sheets from WT mice ( Figure 3D), suggesting an autocrine loop was involved in IL-23 production by keratinocytes.
The role of IL-1 and IL-23 in inducing a protective IL-17A response in S. aureus-infected, mechanically injured skin was examined in Il1r1 -/mice and following IL-23 blockade in WT mice, respectively. Cutaneous Il17a expression, neutrophil infiltration, and S. aureus clearance were significantly decreased in Il1r1 -/mice compared with WT controls as well as in mice treated with neutralizing anti-IL-23p19 antibody compared with mice treated with IgG isotype control (Figure 3, E and F). These results indicate that both IL-1 and IL-23 play an important role in driving IL-17A production by TCRγδ + cells during superficial skin infection. Basophil-derived IL-4 suppresses cutaneous Il17a expression and promotes S. aureus infection of mechanically injured skin. IL-4 impairs the induction and maintenance of adaptive Th17 immune responses (36). We investigated whether IL-4 suppresses the TCRγδ + cell-dependent cutaneous IL-17A response that protects tape-stripped skin from superficial infection with S. aureus. Expression of Il4 was minimal in shaved skin and did not increase following application of S. aureus ( Figure 4A). Tape stripping significantly increased Il4 expression in the skin; exposure to S. aureus increased it further ( Figure 4A). Cutaneous expression of Il17a and of the chemokine Cxcl1 and of the AMP Camp following application of S. aureus to tape-stripped skin was significantly increased in Il4 -/mice compared with WT controls (Figure 4, B-D). Importantly, the numbers of S. aureus CFU recovered from skin homogenates after S. aureus application to tape-stripped skin were significantly decreased in Il4 -/mice compared with WT controls ( Figure 4E).
Basophils are an important source of innate cell-derived IL-4 (38). Flow cytometry analysis of skin cell suspensions revealed that CD45 + CD3 -IgE + CD117basophils accounted for less than 1% of CD45 + CD3cells in shaved mouse skin before and after application of S. aureus. This percentage significantly increased 24 hours after tape stripping and markedly increased following application of S. aureus to tape-stripped skin ( Figure 4F). In contrast, tape stripping and S. aureus application caused minimal changes in the percentages of IgE + CD117 + mast cells in the skin ( Figure 4F). To investigate whether basophils are a major source of  IL-4 in tape-stripped skin, Mcpt8 DTR/+ mice, which express diphtheria toxin receptor (DTR) selectively in basophils (39), and their Mcpt8 +/+ littermates were treated with DT. The percentage of basophils in tapestripped skin exposed to S. aureus was drastically decreased in DT-injected Mcpt8 DTR/+ mice compared with DT-injected Mcpt8 +/+ controls ( Figure 4G). Importantly, Il4 expression in tape-stripped skin sites exposed to S. aureus was significantly diminished in DT-injected Mcpt8 DTR/+ mice compared with controls ( Figure  4H). In contrast, Il17a expression at these sites was significantly higher in DT-injected Mcpt8 DTR/+ mice compared with controls ( Figure 4I). Importantly, the numbers of CFU recovered from skin homogenates after S. aureus application to tape-stripped skin were significantly reduced in DT-injected Mcpt8 DTR/+ mice compared with controls ( Figure 4J).
To investigate the role of basophil-derived IL-4 in S. aureus colonization of mechanically injured skin, we generated  Figure 4K). In addition, basophils from Mcpt8 cre/+ Il4 fl/-13 fl/+ mice were severely impaired in their ability to produce IL-4 compared with basophils from Il4 fl/+ 13 fl/+ control littermates ( Figure 4L). In contrast, cutaneous IL-1α, IL-1β, and IL-23 mRNA and protein levels at these sites were significantly increased in Mcpt8 cre/+ Il4 fl/-13 fl/+ mice compared with controls ( Figure 4, M-O). In addition, Mcpt-8 cre/+ Il4 fl/-13 fl/+ mice exhibited increased cutaneous IL-17A production, due to the increased percentage of IL-17A + TCRγδ + cells infiltrating their infected skin ( Figure 4P). Importantly, the numbers of S. aureus CFU recovered from skin homogenates after S. aureus application to tape-stripped skin were significantly reduced in Mcpt8 cre/+ Il4 fl/-13 fl/+ mice compared with controls ( Figure 4Q). Together, these results indicate that IL-4 derived from basophils recruited to mechanically injured skin promotes cutaneous infection with S. aureus. IL-4 acts at multiple checkpoints to inhibit the protective cutaneous IL-17A response against superficial infection by S. aureus. Mechanical injury induced by tape stripping promotes the production of damage-associated molecular patterns and cytokines. These include TNF-α and hyaluronic acid (HA), which drive the expression of IL-1α and IL-23, respectively, in keratinocytes (33,40). Keratinocytes express type II IL-4 receptor (IL-4R). The type II IL-4R binds both IL-4 and IL-13 and shares the IL-4Rα chain with the type I IL-4R, which binds IL-4 but not IL-13. Addition of rIL-4 inhibited TNF-α-driven Il1a expression as well as HA-driven Il23p19 expression by epidermal sheets from WT mice but not Ilr4a -/mice ( Figure 5, A and B). These in vitro findings suggested that IL-4 could inhibit cutaneous Il17a expression in response to S. aureus exposure by downregulating IL-1α and IL-23 production by keratinocytes. To test this hypothesis, we generated K14-Cre Tg/0 Il4ra fl/mice, which lack IL-4Rα selectively in keratinocytes. Cutaneous expression of Il1a, Il23p19, and Il17a following S. aureus application to tape-stripped skin was significantly increased in K14-Cre Tg/0 Il4ra fl/mice compared with K14-Il4ra fl/controls ( Figure 5, C-E). Importantly, the numbers of CFU recovered from skin homogenates after S. aureus application to tape-stripped skin were significantly reduced in K14-Cre Tg/0 Il4ra fl/mice compared with controls ( Figure 5F).
TCRγδ + cells, the major producers of IL-17A in mechanically injured skin exposed to S. aureus, express type I IL-4R (41). We investigated whether IL-4 has a direct effect on Il17a expression by TCRγδ + cells following stimulation with IL-1 and IL-23. rIL-4 markedly inhibited IL-1β+IL-23-driven Il17a expression by TCRγδ + cells sorted from the skin of WT mice but not Ilr4a -/mice ( Figure 5G).
Keratinocytes are a major target for IL-17A-driven expression of neutrophil-attracting chemokines (42). We examined whether IL-4 has a direct effect on IL-17A-driven expression of the neutrophil chemoattractant Cxcl1 by keratinocytes. rIL-4 inhibited IL-17A-driven expression of the neutrophil-attracting chemokine Cxcl1 by epidermal sheets from WT mice but not Ilr4a -/mice ( Figure 5H). The above results suggest that IL-4 may act at multiple checkpoints to inhibit the protective cutaneous IL-17A response of mechanically injured skin to S. aureus and consequently promote infection.
IL-4Rα blockade protects mechanically injured skin from superficial S. aureus infection. IL-4Rα blockade by mAb is approved for human use. We investigated whether IL-4Rα blockade could protect against S. aureus infection of mechanically injured skin. Mice were administered rat anti-mouse IL-4Rα-blocking mAb or IgG isotype control 2 hours prior to application of S. aureus to tape-stripped skin. Cutaneous expression of Il1a, Il23p19, and Il17a as well as neutrophil infiltration were significantly higher in S. aureus-exposed, tapestripped skin of recipients of the IL-4Rα-blocking antibody compared with recipients of IgG isotype control ( Figure 6, A and B). However, there were no significant changes in Il4 mRNA levels or in the percentages of TCRγδ + cells and basophils following IL-4Rα antibody blockade (Supplemental Figure 3). Importantly, JCI Insight 2021;6(21):e149953 https://doi.org/10.1172/jci.insight.149953 IL-4Rα blockade significantly reduced the numbers of S. aureus CFU recovered from the skin ( Figure 6C). These results indicate that IL-4 signaling blockade restrains S. aureus infection of mechanically injured skin.

Discussion
We have uncovered an important role for basophil-derived IL-4 in promoting superficial skin infection with S. aureus. Our results demonstrate that basophils are recruited following exposure of S. aureus on tapestripped skin and that basophil-derived IL-4 acts at multiple checkpoints to inhibit the IL-17A response of TCRγδ + T cells that protects against infection. Importantly, we show that IL-4Rα blockade, currently in use for the treatment of patients with AD, protects tape-stripped skin from superficial S. aureus infection.
Shaved but otherwise intact mouse skin was resistant to the establishment of superficial skin infection by S. aureus. In contrast, mouse skin mechanically injured by tape stripping was permissive for S. aureus superficial skin infection. Like intradermal and subcutaneous infection with S. aureus, superficial S. aureus skin infection elicited a TCRγδ + cell-dependent protective local IL-17A response that promoted neutrophil recruitment and AMP production, both of which are important for the clearance of S. aureus (7,8,14,43). IL-1 and IL-23, which may be derived in part from keratinocytes, were required for the induction of IL-17A following S. aureus infection of mechanically injured skin, consistent with the important role of these 2 cytokines in the induction of IL-17A in inflammation, infections, and wound healing (16,18,19,34). IL-1 signaling was required for the optimal induction of cutaneous Il23p19 expression in skin superficially infected with S. aureus. This is in line with a recent report indicating that recombinant IL-1R antagonist decreases Il23p19 expression and neutrophil recruitment into the skin induced by ablation of corneodesmosin (44).
We have shown that basophils are recruited to mechanically injured skin exposed to S. aureus and partially block the IL-17A/neutrophil recruitment axis that protects against superficial skin infection. In agreement with this result, acute depletion of basophils exacerbates mouse models of experimental autoimmune encephalomyelitis (EAE) and colitis mediated by IL-17A and neutrophils (26,28,45). Other studies have shown that basophils promote type 17 immune responses by producing IL-6 and that chronic depletion of basophils exacerbates the lupus phenotype in MRL-lpr/lpr mice and the severity of EAE in WT mice (25,  27). Thus, basophils may play a context-dependent dual role in the in vivo IL-17 response, initially suppressing its induction, then once it is established, supporting its maintenance.
Basophils are a key source of IL-4 under steady-state as well as pathological conditions (38). Our results demonstrate that basophils are the main source of IL-4 in skin superficially infected with S. aureus and that basophil-derived IL-4 dampens the protective IL-17A response against infection. This inhibition is in line with the known role of IL-4 in suppressing in type 17 responses. IL-4 suppresses IL-17A expression by CD4 + T cells as well as the in vitro production of the IL-17-inducing cytokines IL-23p19, IL-1β, and IL-6 by DCs and keratinocytes (24,36). In vivo, treatment with rIL-4 improves disease severity in mouse models of EAE and psoriasis, 2 disease models associated with a type 17 immune response (36,46,47).
Keratinocytes have emerged as important players in cutaneous immune responses, as they respond to autocrine and paracrine cytokines by producing cytokines, chemokines, and AMP. We show that IL-4 suppresses Il1a, Il23p19, and Cxcl1 expression by epidermal sheets in response to in vitro stimulation with TNF-α, HA, and IL-17A, respectively. Importantly, mice lacking IL-4Rα selectively in keratinocytes demonstrated increased cutaneous expression of the cytokines Il1a, Il23p19, and Il17a in mechanically injured skin exposed to S. aureus. In line with these results, IL-4 inhibits TNF-α-as well as IFN-γdriven expression of AMP-encoding genes by keratinocytes (48). It also suppresses the production of IL-1β and the AMP hBD2 in epidermal cells from patients with psoriasis (49), who exhibit increased IL-4Rα expression by epidermal cells (50). Together, these results suggest that IL-4 signaling in keratinocytes inhibits cutaneous IL-17A induction and plays a counterpoint to IL-17 signaling in these cells, possibly in an attempt to restore homeostasis in inflamed skin.
The diversity of the skin microbiome and its interactions with pathogenic microorganisms shape the immune response and limit the growth of pathogens (51)(52)(53). We cannot exclude that perturbation of skin microbiome induced by tape stripping or in mice with deficiencies in cells or cytokines could affect the establishment of cutaneous S. aureus infection. Furthermore, different S. aureus strains produce different virulence factors and toxins that could alter the protective immune response differently than the S. aureus SF8300 strain we used.
Administration of dupilumab, a human mAb against IL-4Rα, to patients with AD ameliorates the disease and results in improved local and systemic disease markers, as well as a decrease in S. aureus colonization of the skin (54)(55)(56)(57). It is difficult to dissect the exact mechanisms by which IL-4Rα blockade reduces the S. aureus cutaneous burden in patients with AD. We demonstrate that IL-4Rα blockade increases Il17a expression and promotes bacterial clearance from the skin in our mouse model of acute superficial infection with S. aureus. This is of clinical relevance because it suggests that IL-4R blockade might represent an effective therapeutic approach to inhibit S. aureus skin colonization or prevent S. aureus SSTIs.