Today, I happened to read a very interesting article from the lab of Dr. Llyod S Miller, Division of Dermatology, UCLA, USA. This article shows that IL-17 is essential for host defense against cutaneous Staphylococcus aureus infection in mice. The article is cited as Cho JS, Pietras EM, Garcia NC, Ramos RI, Farzam DM, Monroe HR, Magorien JE, Blauvelt A, Kolls JK, Cheung AL, Cheng G, Modlin RL, Miller LS. IL-17 is essential for host defense against cutaneous Staphylococcus aureus infection in mice. J Clin Invest. 2010 May 3;120(5):1762-73.

I have tried to prepare a brief synopsis of the article and it is as follows:
Staphylococcus aureus is a gram positive bacteria that is one of the causes of skin and soft tissue infections. In recent years, virulent antibiotic resistant strains, such as methicillin resistant S. aureus (MRSA) have become a major cause of public health concern. Immune based therapy may provide an alternative approach for treatment of these infections. The use of immune based therapy requires a clear understanding of the protective immune response against S. aureus infection in the skin. The neutrophils recruitment has been shown to play an essential role S. aureus infection. Other studies have also provided evidence that in addition to neutrophils, T cells also play important roles in host defense against S. aureus cutaneous infection. However, the mechanisms by which T cells promote protective immunity against S. aureus skin infection are not clear. Thus, in the present study the investigators decided to evaluate the contribution and mechanism by which alpha beta or gamma delta T cells participate in cutaneous host defense. For this they used an in vivo mouse model of cutaneous S. aureus infection.

The study was performed in a stepwise manner and these were as follows:

1. First step was to know which T cells contribute to host defense against S. aureus infection in skin. Thus, wild type mice and mice deficient in either alpha beta or gamma delta T cells were inoculated intradermally with the bioluminescent S. aureus SH100 strain. The lesion size and in vivo bioluminescence of the live actively metabolizing bacteria within the lesions were evaluated. It was observed that gamma delta T cell deficient mice developed skin lesions approximately three fold larger than wild type or alpha beta deficient mice. To determine a possible association of large lesions of gamma delta deficient mice with impaired bacterial clearance the authors anesthesized the mice and determined the bacterial counts within the lesions in real time. It was observed that number of CFUs obtained from 8mm punch biopsies were up to 10 fold higher in gamma delta T cells deficient mice than in WT mice on days 1 and 3 of infection. The authors have also earlier observed that after, S. aureus infection, WT and alpha beta T cell deficient mice had similar bioluminescent signals that decreased over 14 days. In contrast, gamma delta T cells deficient mice showed bioluminescent signals that were up to 20 fold higher than those of WT mice at all time points.
All these data indicate that gamma delta T cells, but not alpha beta T cells are required for mediating host defense and bacterial clearance against cutaneous S. aureus infection.


2. The next step was to know the role of gamma delta T cells in recruitment of neutrophils to site of infection. To investigate this, histology and myeloperoxidase (MPO, an indicator of neutrophils function) activity were evaluated in skin lesions biopsies performed after one day on inoculation in WT and gamma delta T cell deficient mice. The lesions of gamma delta T cell deficient mice had highly decreased number of neutrophils with impaired abscess formation and a good number of S. aureus bacteria as observed by Gram staining in comparison to WT mice. The MPO activity was also found t be less in deficient mice. These data suggest that gamma delta T cell deficient mice show impaired recruitment of neutrophils to the site of infection and thus gamma delta T cells play important role in recruitment of neutrophils. Next step was to further investigate the mechanism of impaired neutrophils recruitment observed in gamma delta T cell deficient mice. This was done by assessing the levels of soluble factors which are known to have direct neutrophils chemotactic activity. These included neutrophils chemokines, KC and MIP2 and granulopoeisis factor GM-CFS. The levels were measured by performing real time PCR and protein array analysis on homogenized lesion biopsies 8 hr after inoculation. The gamma delta T cell deficient mice showed significantly decreased levels of mRNA and proteins. The results suggested the impaired neutrophils recruitment in gamma delta T cell deficient mice was most probably caused by a decrease in production of neutrophils chemotactant.

3. The third step was to investigate the production of T cell derived cytokines which are known to promote neutrophils recruitment. These cytokines are IL-17 and IL-22. These cytokines trigger fibroblasts and epithelial cells to produce neutrophils recruitment factors, e.g., KC, MIP2 and GM-CSF. The authors also evaluated production of IL-21. They found that WT mice showed an early gene expression of IL-17A and IL-17F in response to the bacterial challenge (mRNA levels by real time PCR). In contrast, gamma delta T cell deficient mice did not show this early expression of IL-17A and IL-17F. IL-22 was found to be expressed in both groups of mice. The authors then further studied the source of IL-17 in skin sample lesions from WT mice. It was noted that IL-17Aand IL-17F were highly expressed in the epidermal compartment and there was more or less no expression in the dermis. The authors then positively selected the CD3+ from cell suspensions by using a specific monoclonal antibody directed against the T cell marker. It was observed that after 8 hrs of infection, IL-17A and IL-17F induction was detected exclusively in T cell fraction (CD3+ cells). In contrast, no such induction was observed in T cell fractions or the negative fractions from epidermis of gamma delta T cell deficient mice. The authors further selected gamma delta T cells from cell suspensions using gamma delta specific antibody, GL3. IL17A and IL17F induction was detected exclusively in gamma delta T cell fractions from WT mice. All these results indicate that gamma delta T cells are major source of IL-17 production in response to S. aureus cutaneous challenge.

4. The next step was to determine the exact location of gamma delta T cells within the infected skin lesions and the percentage of gamma delta T cells that express the known skin specific Vgamma5 chain. The authors thus examined the sections of lesional biopsy at 8 and 24 hrs after skin inoculation and observed CD3+ cells in epidermal compartment of WT mice. Only rare CD3+ cells were seen in epidermis of gamma delta T cell deficient mice. Further, epidermal cell suspensions of skin biopsies from normal uninfected WT mouse skin were labeled with specific mAbs for CD3, gamma delta T cells and Vgamma5 expressions were analyzed by flow cytometry. 90% of CD3+ T cells from WT mice epidermis were found to express Vgamma5 chain. Purified GL3+ epidermal gamma delta T cells from uninfected mice were left unstimulated or were activated with PMA/ionomycin and cultured in the absence or presence of IL-1beta, IL-23 or both IL-1beta and Il-23. Supernatants were collected after 24 hrs and IL-17 levels were analyzed by ELISA. PMA/ionomycin stimulation resulted in a three fold increase in production of IL-17 compared with unstimulated cells. The addition of exogenous recombinant IL-1beta or IL-23 or combination of both resulted in 2-3.5 fold increase in IL-17 production over PMA/ionamycin alone. All these data indicate that resident epidermal gamma delta T cells expressing Vgamma5 chain are the predominant T cell subset present in mouse skin at baseline and during an early immune response to S. aureus skin infection and they have the capacity to produce IL-17 upon activation.

5. In the next step, the authors determined whether IL-17 production in response to S. aureus infection by gamma delta T cells is dependent up on IL-1, TLR-2 (TLR-2 has been recently shown to be expressed by gamma delta T cells) and IL-23. To determine whether the induction of IL-17A and IL-17F was dependent up on IL-1 and/or TLR-2, the authors inoculated WT mice and mice deficient in IL-1R, TLR-2 or MyD88 and determined IL-17A and Il-17F mRNA production at different time points by real time PCR. At 8 hrs after inoculation, IL-1R-, TLR-2 and MyD88-deficient mice showed impaired expression of IL-17A and IL-17F compared with WT mice. Similarly, to determine the dependence of IL-17A and IL-17F expression on IL-23, the authors inoculated WT mice and mice deficient in p40 (shared subunit for IL-12 and IL-23) and p19 (IL-23 specific subunit) and p35 (Il-12 specific subunit). It was observed that at 8 hrs after challenge p40 and p19 but not p35 deficient mice showed decreased expression of IL-17A or IL-17F in comparison to WT mice. This shows that IL-23 and not IL-12 plays an important role in induction of IL-17A and IL-17F.

6. The further step was to determine whether IL-17 contributes directly to the impaired neutrophils recruitment and host defense against S. aureus cutaneous challenge as seen in gamma delta T cell deficient mice. Thus, WT mice and mice deficient in the IL-17R were inoculated intradermally with S. aureus. It was observed that IL-17R deficient mice developed significantly larger lesions and higher bacterial counts in comparison to WT mice. Thus, these data indicate that IL-17 producing gamma delta T cells are required for host defense against S. aureus skin infections.

7. Given the critical role of gamma delta T cells and IL-17 in host defense against cutaneous S. aureus challenge, the final step was to determine whether the addition of IL-17A (recombinant IL-17) can affect the increased lesion size and bacterial counts in gamma delta T cell deficient mice. Administration of single dose of rIL-17 along with the S. aureus inoculum resulted in significantly reduced lesion sizes and bacterial counts compared with gamma delta T cell deficient mice that were inoculated with S. aureus alone. The lesion size and bacterial counts observed in gamma delta T cell deficient mice that were immunized with rIL-17 were found to be comparable to those observed in wild type mice. These data suggest that IL-17 is the essential mediator by which gamma delta T cells contribute to host defense against S. aureus cutaneous infection.