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A Hairy Tale of Monocytes and Contact Hypersensitivity Reactions

  • Author Footnotes
    2 These authors contributed equally to the work.
    Benjamin Voisin
    Footnotes
    2 These authors contributed equally to the work.
    Affiliations
    Dermatology Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, Maryland, USA
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  • Author Footnotes
    2 These authors contributed equally to the work.
    Thomas Doebel
    Footnotes
    2 These authors contributed equally to the work.
    Affiliations
    Dermatology Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, Maryland, USA
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  • Keisuke Nagao
    Correspondence
    Correspondence: Keisuke Nagao, Dermatology Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bldg 10, Room 12N240B, 10 Center Drive, Bethesda, Maryland, 20892-1908, USA.
    Affiliations
    Dermatology Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, Maryland, USA
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  • Author Footnotes
    2 These authors contributed equally to the work.
      Hair follicles have recently emerged as immunologically active organs that orchestrate recruitment and trafficking of immune cells within skin. Liu et al. (2018) expand our knowledge in this growing area of research by characterizing the network of immune cell interactions during experimental contact hypersensitivity that, interestingly, is centered around hair follicles.
      In aggregate, Liu et al. (2018) provide evidence for an antigen recognition-dependent immunological cascade during CHS, in which HFs seem to mediate the encounter and interaction of immune cells involved.
      Immunity at body surfaces involves an intricate network of immune cell interactions that rely heavily on innate mechanisms initiated in the epithelium. Such interactions are multidirectional and may involve various immune and non-immune cell types of the epithelium and subepithelial layers that facilitate the propagation of effective immune responses. For example, perturbation of skin mediated by mechanical stress, allergens, or microbes may provoke keratinocytes to produce chemokines and cytokines that recruit and activate immune cells. Cytokines that are produced by activated immune cells, such as monocytes/macrophages (TNF), dendritic cells (IL-23), or lymphocytes (IL-17, IL-22) may also act on the epidermis to enhance the production of host-protective soluble factors, such as antimicrobial peptides. Hair follicles (HFs) have recently emerged as immunologically functional structures that mediate skin-specific regulation of the immune system. Interactions that involve HF are also not unidirectional and they may involve positive- and negative-feedback loops, many of which are uncharacterized. We have shown previously that minor mechanical stress leads to the production of CCL2 and CCL20 in the upper regions of HFs. These chemokines then mediate CCR2- and CCR6-dependent recruitment of monocytic precursors of epidermal Langerhans cells (
      • Nagao K.
      • Kobayashi T.
      • Moro K.
      • Ohyama M.
      • Adachi T.
      • Kitashima D.Y.
      • et al.
      Stress-induced production of chemokines by hair follicles regulates the trafficking of dendritic cells in skin.
      ). HFs also support epidermal resident memory T-cell persistence by constitutively producing cytokines IL-7 and IL-15 (
      • Adachi T.
      • Kobayashi T.
      • Sugihara E.
      • Yamada T.
      • Ikuta K.
      • Pittaluga S.
      • et al.
      Hair follicle-derived IL-7 and IL-15 mediate skin-resident memory T cell homeostasis and lymphoma.
      ). Building on their previous work demonstrating that regulatory T cells preferentially localized around HFs (
      • Gratz I.K.
      • Truong H.A.
      • Yang S.H.
      • Maurano M.M.
      • Lee K.
      • Abbas A.K.
      • et al.
      Cutting edge: memory regulatory T cells require IL-7 and not IL-2 for their maintenance in peripheral tissues.
      ),
      • Ali N.
      • Zirak B.
      • Rodriguez R.S.
      • Pauli M.L.
      • Truong H.A.
      • Lai K.
      • et al.
      Regulatory T cells in skin facilitate epithelial stem cell differentiation.
      recently showed that skin-resident regulatory T cells were a source of the Notch ligand, Jagged 1, that promoted HF stem cell proliferation and differentiation. Thus, the follicular microenvironment provides a niche for tissue-immune crosstalk that is crucial not only for immunological homeostasis, but tissue homeostasis as well.
      The work by Liu et al. (2018) expands our knowledge on HF-immune system crosstalk. Utilizing oxazolone (OXA)-induced contact hypersensitivity (CHS), they provide evidence for an antigen-recognition–dependent activation of HF keratinocytes that then produce chemokines to recruit monocytes (Figure 1). The authors began their study by monitoring the spatiotemporal behavior of monocytes during OXA-induced CHS using intravital microscopy. Over the course of 24 hours post OXA challenge, they observed skin infiltration of inflammatory monocytes (characterized by flow cytometry as CX3CR1intLy6ChighCD115+F4/80+CCR2+cells) within the dermis as massive clusters around HFs. Although monocyte recruitment appears to occur at a later time point in OXA-induced CHS compared to tape stripping (
      • Nagao K.
      • Kobayashi T.
      • Moro K.
      • Ohyama M.
      • Adachi T.
      • Kitashima D.Y.
      • et al.
      Stress-induced production of chemokines by hair follicles regulates the trafficking of dendritic cells in skin.
      ), the dynamics of monocyte infiltration was strikingly similar between the two methods of perturbation, pointing to common mechanisms that might drive skin leukocyte trafficking toward HFs after different types of inflammatory stimuli.
      Figure 1
      Figure 1Immunological cascade triggered by oxazolone challenge. During the first 12 hours post skin challenge with oxazolone, pro-inflammatory monocytes are recruited to the dermis. Meanwhile, clusters of T cells and dendritic cells form around hair follicles. Focal oxazolone-specific T cells release IFN-γ that acts on adjacent keratinocytes to trigger chemokine release. During an additional 12 hours, CCL2, together with CXCR2 ligands, mobilize and direct monocytes to accumulate around hair follicles. Once aggregated, monocytes lead to focal keratinocyte apoptosis potentially via TNF-α production.
      To gain more insight into the molecular mechanisms that drove monocyte recruitment, OXA-challenged mice were systemically treated with selected chemokine receptor antagonists. Only antagonism of CCR2 inhibited monocyte accumulation within the dermis to a level comparable to that mediated by pertussis toxin (a pan G-protein–coupled receptor inhibitor), emphasizing the importance of CCR2 signaling during monocyte recruitment. Interestingly, the authors found that monocyte accumulation around the HFs required CXCR2, the receptor for the chemokine CXCL8 (IL-8), a well-established mediator of inflammation in CHS (
      • Kaplan D.H.
      • Igyarto B.Z.
      • Gaspari A.A.
      Early immune events in the induction of allergic contact dermatitis.
      ).
      Given the recently demonstrated importance of T cell–dendritic cell cluster formation mediated by macrophages in CHS (
      • Natsuaki Y.
      • Egawa G.
      • Nakamizo S.
      • Ono S.
      • Hanakawa S.
      • Okada T.
      • et al.
      Perivascular leukocyte clusters are essential for efficient activation of effector T cells in the skin.
      ), the authors adoptively transferred red fluorescent dye–labeled OXA-sensitized T cells into CX3CR1GFP CD11cEYFP mice to define the spatial relationships of T cells, CX3CR1mid monocytes, and dendritic cells via intravital confocal microscopy. Interestingly, OXA challenge induced accumulations of T cells and dendritic cells around HFs after 12 hours. These cells were joined by monocytes after an additional 12 hours. Importantly, OXA challenge in mice that were sensitized with the hapten, 2,4-dinitrofluorobenzene, did not result in monocyte accumulation around HFs, indicating that T cells reactive to OXA-modified antigens were required for this process. The authors then hypothesized that IFN-γ produced by antigen-specific T cells might act as a soluble mediator that promoted the accumulation of monocytes around HFs. To address this, they adoptively transferred presensitized wild-type or IFN-γ–deficient T cells into wild-type mice and compared monocyte cluster formation after OXA treatment. Monocytes still formed clusters around HFs, but their numbers were markedly decreased in mice that received lymph node cells from OXA-sensitized IFN-γ–deficient mice. One caveat in this experiment is that lymph node cells collected from OXA-sensitized IFN-γ–deficient mice may not have contained comparable numbers of OXA-specific effector T cells. Because comparable T cell accumulation to HFs was not demonstrated in these mice, it remains to be determined if reduction of monocyte cluster formation is a direct consequence of IFN-γ deficiency in skin T cells. Nevertheless, CXCR2 ligands were not increased in these mice, and real-time PCR analysis on enriched Sca1 HF keratinocytes demonstrated decreased expression of mRNA encoding CCL2. Taken together, the authors propose that IFN-γ produced by OXA antigen-specific T cells, presumably TH1 and/or CD8+ T lymphocytes, triggers the release of chemokines by HF keratinocytes that subsequently attract monocytes.
      Taking advantage of CXCR2-dependent clustering process, the authors then profiled the activation state of clustered monocytes in OXA-challenged mice that were either untreated or pretreated with CXCR2 antagonists and showed that aggregation of monocytes was a prerequisite for TNF-α production. Given the pro-inflammatory properties of TNF-α and its ability to induce apoptosis (
      • Varfolomeev E.
      • Vucic D.
      Intracellular regulation of TNF activity in health and disease.
      ), monocyte accumulation at the dermal–epidermal junction of HFs could be detrimental to tissue homeostasis. Indeed, the authors observed an increased ratio of apoptotic HF keratinocytes at sites of monocyte accumulation. Interestingly, keratinocyte apoptosis was decreased when monocytes were depleted via systemic clodronate injection 24 hours prior the OXA challenge. Moreover, inhibition of monocyte clustering by CXCR2 antagonist administration was also associated with significant reduction in keratinocyte apoptosis, indicating that focal accumulations of monocytes around HFs leads to keratinocyte apoptosis. Although the significance of keratinocyte apoptosis in CHS is still unclear, the depletion of monocytes by clodronate injection in this CHS model eventually decreased ear swelling, suggesting a broader role of monocytes in the inflammatory process following OXA challenge. Perhaps the monocyte-mediated keratinocyte apoptosis described in this report is analogous to collections of Langerhans cells that can be observed in intra-epidermal vesicles in allergic contact dermatitis in humans (
      • Rosa G.
      • Fernandez A.P.
      • Vij A.
      • Sood A.
      • Plesec T.
      • Bergfeld W.F.
      • et al.
      Langerhans cell collections, but not eosinophils, are clues to a diagnosis of allergic contact dermatitis in appropriate skin biopsies.
      ). It may also be important to note that apoptosis is not a described feature in allergic contact dermatitis in humans.
      In aggregate, Liu et al. (2018) provide evidence for an antigen recognition-dependent immunological cascade during CHS, in which HFs seem to mediate the encounters and interactions of immune cells that are involved. Several interesting questions that were raised by the study remain to be addressed in future work. For example, the authors observed that T cell–dendritic cell cluster formation around HFs after OXA challenge preceded monocyte accumulation. Previous work in a similar CHS model described macrophage-dependent formation of perivascular T cell–dendritic cell clusters (
      • Natsuaki Y.
      • Egawa G.
      • Nakamizo S.
      • Ono S.
      • Hanakawa S.
      • Okada T.
      • et al.
      Perivascular leukocyte clusters are essential for efficient activation of effector T cells in the skin.
      ). It would be interesting to see how the vasculature relates to the clusters observed in the OXA-dependent CHS model and whether macrophages, in addition to monocytes, play a role in cluster formation. Another important aspect of the study that remains to be addressed is whether monocytes directly mediate keratinocyte apoptosis. The observation by Liu et al. (2018) that IFN-γ, a cytokine critical for cytotoxic responses, is only involved in recruiting monocytes is rather counter-intuitive. Accumulation and production of TNF-α by monocytes correlated with keratinocyte apoptosis, but direct involvement of monocyte TNF-α remains to be shown.
      Clusters of myeloid cells around HFs have been described in different contexts. Even under physiological conditions, myeloid cells associate with HFs and are thought to mediate destruction of HFs during the hair cycle (
      • Eichmuller S.
      • van der Veen C.
      • Moll I.
      • Hermes B.
      • Hofmann U.
      • Muller-Rover S.
      • et al.
      Clusters of perifollicular macrophages in normal murine skin: physiological degeneration of selected hair follicles by programmed organ deletion.
      ). Both OXA challenge as described by
      • Liu Z.
      • Yang F.
      • Zheng H.
      • Fan Z.
      • Qiao S.
      • Liu L.
      • et al.
      Visualization of T cell-regulated monocyte clusters mediating keratinocyte death in acquired cutaneous immunity.
      and mechanical stress after gentle tape stripping induce CCL2 production (
      • Nagao K.
      • Kobayashi T.
      • Moro K.
      • Ohyama M.
      • Adachi T.
      • Kitashima D.Y.
      • et al.
      Stress-induced production of chemokines by hair follicles regulates the trafficking of dendritic cells in skin.
      ) that lead to the recruitment of monocytes. Therefore, CCL2 production by HF keratinocytes could be a common response after various insults that involve the epithelium, a response that is then fine-tuned by additional context-dependent factors. If applicable to human allergic contact dermatitis, pathways involving CCL2 may be attractive therapeutic targets. The source of CXCR2 ligands that attract monocytes to the HFs has not been clarified. As more and more data implicate HFs in different immunological contexts, it is interesting to speculate that HFs might have a much broader role than previously appreciated as potential orchestrators of skin immune responses.

      Conflict of Interest

      The authors state no conflict of interest.

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      Linked Article

      • Visualization of T Cell-Regulated Monocyte Clusters Mediating Keratinocyte Death in Acquired Cutaneous Immunity
        Journal of Investigative DermatologyVol. 138Issue 6
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          It remains unclear how monocytes are mobilized to amplify inflammatory reactions in T cell-mediated adaptive immunity. Here, we investigate dynamic cellular events in the cascade of inflammatory responses through intravital imaging of a multicolor-labeled murine contact hypersensitivity model. We found that monocytes formed clusters around hair follicles in the contact hypersensitivity model. In this process, effector T cells encountered dendritic cells under regions of monocyte clusters and secreted IFN-γ, which mobilizes CCR2-dependent monocyte interstitial migration and CXCR2-dependent monocyte cluster formation.
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